Genes coding proteins for early liver development and their use in diagnosing and treating liver disease

ABSTRACT

Early developing stage-specific liver proteins and the genes coding for them that have been isolated and sequenced are provided, and these genes and proteins can be utilized to diagnose and/or treat a wide variety of liver disorders and other ailments. Included in the proteins identified and isolated in the present invention are the proteins known as elf 1-3, liyor-1 (145), pk, protein 106, and praja-1, along with the nucleic acid sequences coding for these and other proteins, and antibodies, such as the antibodies to mouse elf protein, may be raised which recognize these proteins or to peptides derived from these proteins. Since the early developing liver proteins of the invention arise during embryogenesis when the liver and other organs are in transition from an undifferentiated state to a differentiated one, these proteins are involved in tissue differentiation and thus can be utilized in methods of diagnosing and treating a variety of liver diseases and other disorders including these relating to oncogenesis and tissue repair. Accordingly, the isolated early developing liver proteins in accordance with the present invention should have implications for diagnosis and treatment of a range of diseases from end stage cirrhosis to hepatocellular carcinoma and many other disease conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT application PCT/US98/08656, filed Apr. 30, 1998, which is a continuation-in-part of U.S. patent application Ser. No. 08/841,349, filed Apr. 30, 1997 now U.S. Pat. No. 5,955,594.

FIELD OF THE INVENTION

This invention relates to peptides and proteins isolated during early liver development, genes coding for these peptides and proteins, and antibodies raised to these proteins, and to methods for their use in diagnosing and treating liver disease and other disorders.

BACKGROUND OF THE INVENTION

In the United States and other countries, end stage liver disease due to infection, genetic defects or alcoholic consumption is a major cause of widespread morbidity and mortality, causing great potential hardship and economic loss to millions of people throughout the world. In addition, numerous other diseases, including biliary problems and blood disorders, are associated with disruptions in the many functions carried out by the liver, including iron transport, hepatocyte formation and hematopoiesis. In general, severe problems associated with a breakdown of liver function are practically untreatable, and require a liver transplant as the only cure. However, in light of the great disparity between the number of patients needing liver transplants and the number of donors, thousands upon thousands of people are denied this operation, and transplantation is at the present time not a practical approach to the problem.

At the same time, the precise nature of liver development and the role of early developing liver proteins has not been well understood. To date, no growth factors specific to the liver have been identified or isolated, and the precise molecular mechanisms behind hepatocyte (liver cell) formation remain to be elucidated. There thus has been a long felt need to identify and understand the changes in gene regulation and expression in the developing liver, including the determination as to which genes are switched on and off as a hepatocyte forms and a liver develops. Accordingly, isolating and identifying the genes and proteins which play critical roles in early liver development would be beneficial in understanding the effect of gene regulation and expression in the differentiating liver, and in diagnosing and treating many diseases states involving the liver and liver functions.

SUMMARY OF THE INVENTION

Accordingly, it is thus an object of the present invention to provide genes comprising the nucleic acid sequences encoding early liver developmental proteins, including the liver proteins known as elf 1-3, liyor-1 (145), pk, protein 106, and praja-1.

It is further an object to provide isolated and purified early developing liver proteins encoding by the above genes.

It is still further an object to provide proteins which are characteristic of early liver development and peptides from said proteins and peptides, and to raise antibodies from said proteins and peptides which will be useful as markers, and will be useful in methods of identifying such peptides and proteins, tracing hepatocyte lineage, and treating liver disease.

It is still further an object to use the early developing liver proteins of the present invention to provide liver-specific growth factors for application in diagnosis and treatment of liver disorders.

It is still further an object to provide methods of diagnosing and treating end stage liver disease using the early developing liver proteins of the present invention.

It is even further an object to provide methods of diagnosing and treating other liver disorders and other diseases, including carcinoma, degenerative neurological disorders, anemia, and ataxia, using the early developing liver proteins of the present invention.

These and other objects are achieved by virtue of the present invention which provides genes coding for various proteins which are involved in the differentiation of the developing fetal liver, including the proteins known as elf 1-3, liyor-1 (145), pk, protein 106, praja-1, and a number of other stage-specific genes coding for early-developing liver proteins, and methods for their use in diagnosis and treatment of a variety of liver diseases and other disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in detail with respect to preferred embodiments thereof, which are to be taken together with the accompanying drawings, wherein:

FIGS. 1A-1B represent the nucleic acid sequence encoding the liyor-1 (145) protein in accordance with the present invention.

FIGS. 2A-2E represent the nucleic acid sequence encoding the elf-1 protein in accordance with the present invention.

FIGS. 2F-2I represent the nucleic acid sequence encoding the elf-2 protein in accordance with the present invention.

FIG. 2J represents the nucleic acid sequence encoding the elf-3 protein in accordance with the present invention.

FIGS. 3A-3B represent the nucleic acid sequence encoding the praja-1 protein in accordance with the present invention.

FIGS. 4A-4B represent the nucleic acid sequence encoding the pk protein in accordance with the present invention.

FIG. 5 represents the nucleic acid sequence encoding the 106 protein in accordance with the present invention.

FIGS. 6A-6B represent the nucleic acid sequence encoding gene 20 in accordance with the present invention.

FIG. 7 represents the nucleic acid sequence encoding gene 36 in accordance with the present invention.

FIG. 8 represents the nucleic acid sequence encoding gene 41 in accordance with the present invention.

FIG. 9 represents the nucleic acid sequence encoding gene 112 in accordance with the present invention.

FIG. 10 represents the nucleic acid sequence encoding gene 114 in accordance with the present invention.

FIG. 11 represents the nucleic acid sequence encoding gene 118 in accordance with the present invention.

FIG. 12 represents the nucleic acid sequence encoding gene 129 in accordance with the present invention.

FIG. 13 is a depiction of the membrane skeleton of the ELF protein of the present invention.

FIG. 14 is a graphic representation of known alternatively spliced patterns found among elf transcripts.

FIG. 15 represents ELF expression in primary biliary cirrhosis.

FIG. 16 is a schematic view of the role of SMAD proteins as intracellular mediators of TGF-β and activins.

FIG. 17 depicts α-feto protein labeling cells of hepatocytic lineage in wild type vs. smad2^(+/−)/smad3^(+/−).

FIG. 18 depicts apoptosis in smad2 and smad3 mutants.

FIG. 19 depicts rescue of the hepatic phenotype by culturing in the presence of HGF.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, early developing liver proteins and the genes coding for them have been isolated and sequenced, and these genes and proteins can be utilized to diagnose and/or treat a wide variety of liver disorders and other ailments. In general, the present invention arose from the investigation of liver formation during embryogenesis when the liver and other organs are in transition from an undifferentiated state to a differentiated one. This setting captures the phases of liver formation beginning with ordinary sets of endodermal cells. In addition, the early steps in tissue differentiation are closely related to the process of oncogenesis and tissue repair, and thus the isolated early developing liver proteins obtained in accordance with the present invention should have implications for diagnosis and treatment of a range of diseases from end stage cirrhosis to hepatocellular carcinoma and many other disease conditions.

In the identification and isolation of the liver proteins of the present invention which are useful in early hepatocyte formation, the first step that was taken was to “capture” and analyze gene expression at different stages of early liver formation, particularly at those stages that emerge in the range of about days 9 through 14.5 in the mouse. In this regard, four embryonic liver cDNA libraries were constructed, such as at days 10.0, 11.5, 12.5 and 14.5 post coitus, and after subtractive hybridization, isolation of a group of stage-specific, liver restricted clones were isolated. As will be set forth in more detail below, sequence analysis has revealed that these clones encode a series of early developing liver proteins, which are generally “stage specific”, i.e., they appear only at specific stages of development and not other stages, including elf proteins 1-3, liyor-1 (145), pk, protein 106, proteins coded for by genes 20, 36, 41, 112, 114, 118 and 129, and praja-1, as will be described further herein.

The initial project to identify and isolate developing liver proteins had four main objectives: (1) to construct early embryonic liver libraries; (2) to screen and characterize these early embryonic liver libraries with a group of probes comprising known growth factors (IGF-I, IGF-II, and IGFBP-2) and transcriptional activators (C/EBP and LFB1), known to be expressed in the developing liver; (3) to carry out subtractive hybridization utilizing these cDNA libraries and analyze subsequent subtracted clones for stage specificity by southern blot hybridization, sequence, transcript size, abundance, and tissue distribution; and (4) to develop a functional assay for these subtracted genes using embryonic liver explant cultures.

With regard to the main objectives of the invention, it was decided to focus on the four stages of liver development, particularly around days e10, e11, e12 and e14 (embryonic days post coitus) in developing mice. These four stages are defined developmental time points representing phases of liver development from undifferentiated mesodermal/endodermal cells to a well developed and differentiated fetal liver. These stages have generally been categorized as follows: (1) at around e9-10, a change in cell polarity occurs; (2) at around e10.5-11, invasion and migration of endodermal cells into surrounding mesenchyme occurs; (3) at around e11.5-12, pseudolobule formation, cords of hepatocytes form together with early sinusoids; and (4) at around e12.5-e14.5, the liver is marked by hematopoietic foci and fully differentiated fetal hepatocytes. cDNA libraries representing these stages would therefore represent “captured” mRNA species expressed in greater abundance during critical time periods for hepatocyte formation, enabling their isolation and providing a method for analyzing the changing pattern of gene expression during liver development.

Another aspect of the present invention is the development of useful methods of diagnosis and treatment of liver disorders and other diseases made possible by the identification and isolation of the genes for early developing liver proteins of the invention and the expression of those genes. In accordance with the investigations made regarding these early developing liver proteins, it is clear that the different genes and proteins identified are important for different aspects of liver development and can thus be utilized in treatments of the appropriate disease. During embryogenesis, the liver generally develops from a foregut diverticulum, and comprises four main cell types: the first is the hepatocyte, or endodermal lineage; the second are biliary tree canalicular cells or bile duct cells, the third are hematopoietic cells, and the fourth are the Kupffer cell/Ito cells. As will be set forth below, of the early developing proteins isolated and obtained in accordance with the present invention, it is believed that the elf proteins are important for the formation of the biliary tree, as shown by antisense experiments; praja-1 appears to be important for iron transport and essential for hepatocyte formation as well as hematopoiesis; liyor-1 (145) and pk appear to be important in Ito cell formation and fibrosis.

Accordingly, in accordance with the invention, it is contemplated that elf proteins 1-3 will be useful in the treatment of disorders such as cholestasis, biliary stones, hepatic obstruction, stricture, primary biliary cirrhosis and primary sclerosing cholangitis. In addition, the proteins praja-1, liyor-1 (145) and pk will be useful in treating end stage liver disease, anhidrotic ectoderm dysplasia, hepatocellular carcinoma, as well as anemia, such as sideroblastic anemia, ataxia, e.g., spinocerebellar ataxia, degenerative neurological disorders, anhidrotic ectoderm dysplasia, and hemochromatosis.

Even further, it has also been discovered that the protein praja-1 has been identified in cancerous colon tissue, which normally does not produce this protein. Accordingly, it is contemplated that in accordance with the present invention, a method of detecting and diagnosing colon cancer is provided wherein colon cells or tissues are taken from a patient being tested, and these cells or tissues are screened to determine the presence or absence of the praja-1 protein. Identification of praja-1 in the colon cells or tissues will allow for a determination of whether the cells are cancerous since praja-1 will generally not be detectable in non-cancerous colon cells.

In the preparation of cDNA libraries in conjunction with this invention, it was necessary to utilize the four developmental stages discussed above in order to isolate key early developing liver proteins that affect the formation of hepatocytes and the liver. Although these studies were performed on mice, the relevance of the stages of liver formation to human development is shown in the following summary of these investigations:

(1) Day 10 Post Coitus (e10, 34-39 Somites) (Human Day 27):

In the mouse, the primary liver diverticulum appears during the 10th day of gestation. It develops from a foregut indentation in the endoderm which arises at e7, at the boundary between the embryonic and extraembryonic region, anterior to the developing heart rudiment. At this stage, although the cells are committed to the formation of fetal hepatocytes, they are still epithelial in nature and the liver diverticulum is not viable in the absence of the surrounding heart mesenchyme. As this is the earliest stage possible when hepatocytes are undifferentiated, it was considered to be of great importance: some cells are poised to differentiate into hematopoietic cells and others into hepatocytes. Accordingly, a day 10.0 library was constructed in lambda Unizap, and no prior group had ever constructed a cDNA liver library at this stage.

(2) Day 11.5 Post Coitus (e11.5, 40-44 Somites) (Human Day 32):

This stage is characterized by rapid growth of the liver. Soon after the formation of the hepatic bud, the endodermal cells proliferate, disrupting the membrane separating the epithelium from the septum transversum, with the epithelial cells migrating into the mesenchyme. The liver at e11.5 consists of broad hepatic cords separated by large sinusoids containing nucleated erythrocytes. Hematopoietic foci are found intermingled with the hepatic cords. A cDNA library was constructed in lambda gt10 and lambda Zap from embryonic livers obtained at this stage, since although cells are proliferating rapidly, they still have not attained a fully differentiated fetal state.

(3) Days 12.5-13.0 Post Coitus (e12.5, Human Days 35-45) (Embryo Size: 7-9 mm):

This stage is easily recognized by early signs of finger development as well as by the anterior indentation of the footplate. At this stage, the liver is well developed, all lobes being clearly visible; it contains many megakaryocytes as well as cells with erythropoietic activity. A cDNA library at e12.5 was constructed in lambda gt10 and lambda Zap as this was the earliest stage where fully differentiated fetal hepatocytes are seen.

(4) Day 14.5 Post Coitus (e14.5, Human Days 51-57) (Embryo Size: 20-32 mm):

At this stage, individual, separated forefoot fingers can be seen; hair follicles in the skin can be recognized and the umbilical hernia is very conspicuous. This stage represents a well differentiated fetal liver containing scattered blood-forming foci. A cDNA library of this stage was constructed in lambda Unizap in order to facilitate subtraction with the day 10 library which was also constructed in lambda Unizap (Stratagene).

(5) Adult Mouse Liver:

At birth, day 19, there is a major “switch” in the expression of a large number of genes. From now until the stage at which adult liver is formed, enzyme synthesis of the urea cycle and gluconeogenesis are upregulated. Adult liver is no longer a center for hemopoietic activity except in pathological situations and hepatocytes do not enter de-differentiated states, though the liver still has regenerative capacity as seen in partial hepatectomy.

In conjunction with each of these stages of development, RNA was recovered from each stage, and the quality of the RNA obtained following dissection was assessed by Northern blot analysis using mouse Beta Actin from the Chiba Cancer Center Research Institute, Chiba, Japan. Table 1 shows the RNA yields obtained. The cDNA library construction at days e11.5 and e12.5 of the embryonic liver was carried out by conventional techniques, and the libraries of the day e10.0 and adult mouse liver were obtained using the Stratagene Unizap cDNA library kit. RNA yields and cDNA library size fractionation are shown in Tables 1 and 2, below.

TABLE 1 RNA yields obtained for each stage: TOTAL RNA POLYA + RNA STAGE NO. OF LIVERS (per embryo) (% total RNA) e10.0 608 63 mg (1.04 mg) 26 mg (4%) e11.5 48 60 mg (6.7 mg) 16.8 mg (3%) e12.5 28 N.D. 40 mg (approx.) e4.5 20 N.D. N.D.

TABLE 2 Early embryonic liver cDNA libraries and adult mouse liver library. SIZE AFTER AMPLIFICATION OF LIBRARIES INITIAL SIZE 2 × 106 CLONES VECTOR e10.0 Unizap 6.1 × 106 2.0 × 1010 Lambda Unizap 5.2 × 106 1.6 × 1010 Lambda Zap e11.5 4.1 × 107 2.0 × 1011 Lambda gt10 e12.5 8.0 × 106 1.6 × 1011 Lambda Zap e14.5 1.6 × 107 4.0 × 109 Lambda gt10 Unizap 7.0 × 106 2.0 × 1011 Lambda Adult Unizap 5.0 × 106 2.1 × 1010 Lambda

The cDNA inserts for e10.5, e11.5, e12.5, e14.5 post coitus stage mice and the adult mouse liver were size selected on a Biogel A150 column (>500 bp) prior to ligation to the vector. Actin frequencies for the libraries are given in Table 3.

Qualitative analysis of cDNA libraries utilizing known developmentally regulated cDNAs were carried out in order to establish developmental profiles of important “early” genes that are significant in development, and these libraries were then screened with a specific number of probes. The following probes were obtained and used for screening these libraries, including: Insulin like growth factor I (IGF I), obtained from Dr. Derek le Roith (NIH); Insulin like growth factor II (IGF II) and IGF II binding protein −2(BP-2) both obtained from Dr. Matt Rechler of NIH; LFB 1 obtained from Drs. Monaci, Nicosia and Cortese of EMBL in Heidelberg, and the C/EBP probe from Dr. Darnell of the Rockefeller University in New York, N.Y.

TABLE 3 Clone frequencies for day 10.0, 11.5 and 12.5 libraries (Carried out on Duplicate Screens): Positive cDNA clones per 100,000 poly A + containing cDNA clones. Probe e 10.0 e 11.5 e 12.5 Adult IGF I N.D. Nil Nil N.D. IGF II 3 0 4 0 BP 2 1 1 0 7 LFB I Nil Nil 1 N.D. C/EBP N.D. 2 5 N.D. Beta Actin 120 130 246 270 N.D. = Not Done

The data shows that IGF I was not detected in any of the embryonic libraries, while IGF II was detected in increasing clone frequency from e6.5 to 8.5 (8 at e6.5, 8 at e7.5 and 38 at e8.5—data not shown) and was also detected in the e10.0 and e12.5 libraries (3 at e10.0 and 4 at e12.5—see Table 3). IGF II was not detected in the adult liver library. Interestingly, BP2 clone frequencies are similar to IGF II in the early e6.5, e7.5 and e8.5 libraries (data not shown), but in the liver cDNA libraries the clone frequencies differed, for BP2 only one clone per 100,000 being detected at e10.0 and e11.5, while 7 were detected in the Adult Liver Library compared to the greater numbers for IGF 11. This implied that its temporal and spatial expression in the embryo and fetus is different from IGF 11 and this was subsequently confirmed by in situ studies. LFB I was detected in the e12.5 library, but at one clone per 100,000 screened, which implied that it is not present in mitogenic cells, but that its level was regulated and increased from birth onwards. C/EBP was not present in the e6.5, e7.5, e8.5 or e10.0 libraries (data not shown) but was suddenly detected at day e11.5 and e12.5 in low abundance (about 2 clones/100,000 at e11.5 and 5 at e12.5), implying that while it is expressed, its level also may be regulated, albeit downward, in embryonic stages. Lastly, Beta Actin was used as a reference: all seven libraries had similar Beta Actin frequencies from 120-300/100,000 clones which is considered representative of such embryonic libraries.

Next, identification of stage specific clones by subtractive methods was carried out, and two subtracted libraries were then constructed. Two rounds of subtraction were carried out, and the resulting subtracted libraries comprised 64 clones (e11.5-12.5), and 174 clones (e10.5-14.5) Further characterization of these clones was carried out as follows: (1) Southern hybridization; (2) sequencing; (3) Northern analysis; (4) Zoo blot analysis; and (5) In vitro translation of protein.

In the Southern blotting of these clones, thirty-four clones were shown to be stage specific and not containing mitochondrial, ribosomal and globin sequences. DNA sequencing of these thirty-four stage specific clones was carried out in order to identify clones bearing homology to known developmental genes (such as cell polarity genes, homeobox genes, etc.), and the first 400 base pairs of each clone were sequenced. A detailed analysis was then carried out with respect to some of the clones which form a part of the present invention, including liyor-1 (145), protein 106, pk, and praja-1, since these clones exhibit true stage specificity and appear to belong to a set of genes encoding signal transduction proteins, which are of great interest in development currently, due to studies demonstrating their importance in cell lineage. Other stage-specific proteins which are coded for by genes in accordance with the invention are discussed further below. Studies carried out with regard to proteins such as praja-1 and elf, as well as other early developing liver proteins, have elucidated the sequence of these proteins, as will be set forth in more detail below.

As an example of the tests used to elucidate the developmental expression of these liver proteins, the protein liyor-1 (145) was tested to determine whether these transcripts are differentially expressed during development, specific for mesoderm or endoderm derived tissues, or are expressed in adult mouse and human organs. Accordingly, tissues from mid-gestational embryos were analyzed to determine the role of 145 in liver development. In these tests, tissues were dissected from day 11 onwards, as it was at this stage that discrete hepatic, cardiac and other tissues could be dissected with ease, with subsequent RNA isolated being of good quality.

RNA hybridization with liyor-1 (145) DNA in different mouse tissues was studied by using polyA RNA obtained at various developmental stages using a 32P-labeled 1.1 Kb insert representing protein 145. The specificity of the developmental changes in the steady state levels of 14.5 was evaluated by also measuring the relative levels of Actin. This revealed a 2.4 Kb transcript at high stringency washes. Scanning densitometry of the respective bands revealed that maximal expression of 145 occurred in liver and heart, less so in other tissues, but specifically on day 11 and in decreasing abundance at days 12.5 and 14.5 (when Northerns were developed 1-2 months later).

Further characterization of the distribution of protein 145 RNA in adult tissues and its conservation in evolution has involved RNA analysis of adult mouse and human tissues. The protein 145 hybridizes to adult liver, kidney and testis as a 2.4 Kb transcript in liver and kidney and a 2.6 Kb transcript in adult testis, in very low abundance: both blots were developed after being exposed to film for over a month at −70° C. Similar tests conducted with regard to the elf protein and the nucleic acid coding for it showed that elf DNA is generally conserved across many different species, including human, monkey, rat, mouse, dog, cow, chicken and yeast, and is represented in all species studied except rabbit.

Finally, in accordance with the invention, a functional assay was established for subtracted genes with the goal to establish mouse embryonic liver explant cultures in the laboratory, as this is usually considered the major hurdle for antisense experiments due to the need to dissect extremely small tissue sections at day 9.5 when the liver bud is 0.2 mm. In this regard, the interactions of the neighboring cardiac mesoderm and foregut endoderm were studied and the subsequent changes in cell type specific gene expression were characterized, particularly with respect to alpha-fetoprotein and albumin expression, and partially with respect to epithelial basement membrane components. Methods of culturing liver explants in accordance with the invention are described below. The results obtained in these tests have shown that when cultured in the complete absence of mesodermal derivatives, hepatic endoderm deteriorates rapidly. Only 2 out of 15 such liver explants survived. Hematoxylin and eosin staining showed a necrotic endoderm with no apparent signs of hepatic differentiation. When associated with the surrounding mesoderm particularly cardiac mesoderm (en bloc dissections), the endodermal cells had proliferated and invaded the mesodermal strands. Hepatocytes were seen to be organized in cords separated by sinusoids with pseudo-lobule formation. All 15 out of 15 cultures from en bloc dissections were completely viable. These studies confirm prior explant studies demonstrating the necessity of surrounding mesoderm for liver formation.

Accordingly, cDNA libraries have been constructed for the four main stages of liver development, e10, e11.5, e12.5, e14.5 and for adult liver in the mouse. These have been shown to be truly representative of their respective MRNA species, by meticulous analysis utilizing initial RNA blot analysis, size fractionation, quantitative, and qualitative analysis. Northern analysis confirmed the stage specificity, and restricted expression of their transcripts: for 145 this comprised a 1.35, and 2.37 Kb transcript restricted to midgestational brain and liver tissue, and adult mouse and human Northern blot analysis revealed 145 transcripts in extremely low abundance in liver, kidney, testis. Further tests with regard to protein 145 reveals its sequence identity of 53% (20 S.D. 's) to rat Phospholipase C-γ (PLC-γ), and amino acid alignment of conserved section of 145 to PLC-γ identifies a split pleckstrin homology (PH) domain. Protein 145 (liyor-1) bears 99% identity at the amino acid level to the PH domain at the amino terminus of PLC-γ. The PH domain is an area of 100 amino acids that has been found in a number of proteins including serine/threonine kinases, GTPase activating proteins, phospholipases and cytoskeletal proteins, and is thought to be involved in signal transduction. Nuclear magnetic resonance spectroscopy has revealed that the PH domain of P-fodrin is an electrostatically polarized molecule containing a pocket which may be involved in binding of a ligand. Of immense interest is the fact that this pocket is related to the peptidyl-prolyl-cis-trans-isomerase FKBP in which this pocket is involved in the binding of the macrocyclic compound FK506. Accordingly, it is contemplated that protein 145 may indeed bear a pocket for ‘natural’ ligand similar to FK506 and thus appears to be a potential factor for hepatocyte differentiation.

PLC-γ is regulated by a combination of SH2-domain dependent complex formation with tyrosine phosphorylated receptor tyrosine kinases, and its subsequent phosphorylation on tyrosine residues. An unique feature of PLC-γ and protein 145 is that both contain a split PH domain, which in the case of the PLC fills the gaps between the SH2-SH2-SH3 region and the surrounding X and Y catalytic domains. The SH2 domains mediate the high affinity interaction of PLC-γ with activated growth factor receptors such as epidermal growth factor (EGF) or platelet derived growth factor (PDGF) receptor. The PH domain similarly may be utilized as a specialized noncatalytic domain directing complex formation between protein kinases and their presumptive targets during liver development. In addition, the area of complete identity and split PH domain in 145 and PLC-γ is conserved in a number of other proteins through to TOR2, an essential yeast PI 3 kinase, and to v-abl. A parallel can be drawn to the SH2 domain: that proteins associating with activated growth factor receptors have quite distinct enzymatic properties, are structurally unrelated within their catalytic domains, yet contain a similar noncatalytic domain of approx 100 amino acids, called the src homology (SH) region 2. The SH2 domain was first identified in non receptor protein tyrosine kinase like Src and Fps, by its apparent ability to interact with the kinase domain and phosphorylated substrates. It is believed that during the evolution of cellular signaling mechanisms, the acquisition of SH2 domains conferred on PLC-γ and GAP the capacity to interact with transmembrane tyrosine kinases and therefore couple growth factor stimulation to PI turnover and the kinase pathway. PH domains are similarly conserved and may be utilized in the same way that SH2 domains are.

As indicated above, the protein liyor-1 (145) appears to be important in Ito cell formation and fibrosis, and is thus thought to be useful in treating end stage liver disease as well as other conditions including hepatocellular carcinoma, anemia, ataxia, and hemochromatosis. It is contemplated that the use of the protein Liyor-1 will be by administering to a suitable patient an amount of this liver protein effective to treat the specific condition of that patient, and this would be carried out using conventional means and regimens well known to one skilled in this art. The sequence of Liyor-1 which was determined using the cDNA libraries of the present invention is shown in FIGS. 1A-1B, and suitable amounts of the liyor-1 (145) protein may be prepared in a conventional manner by expressing by recombinant or other means the nucleic acid coding for the 145 protein, after which the protein can be isolated and/or prepared into substantially pure form as needed. In addition, the 145 protein may be administered with any other suitable compound normally utilized for administration into a patient, such as a suitable pharmaceutically acceptable carrier.

As indicated hereinbelow in the examples, other genes for early developing liver proteins in accordance with the present invention have been isolated and sequenced, including the genes coding for the elf proteins, praja-1, pk protein, protein 106, and genes 20, 36, 41, 112, 114, 118 and 129. With regard to the elf proteins, these proteins were studied by analyzing mRNA from tissues from mid-gestation embryos. Tissues were dissected from day 11 onwards, as it was at this stage that discrete hepatic, cardiac and other tissues could be dissected with ease, and the subsequent RNA that was isolated was of good quality. RNA hybridization with elf DNA in different mouse tissues was studied by using polyA RNA obtained at various developmental stages using a ³²P-labeled 1.1 Kb insert representing elf. The specificity of the developmental changes in the steady state levels of elf was evaluated by also measuring the relative levels of Actin. This revealed a 2.4 Kb transcript at high stringency washes. Scanning densitometry of the respective bands revealed that maximal expression of elf occurred in liver and heart, less so in other tissues, but specifically on day 11, and in e12.5 and e14.5 in decreasing abundance (when Northerns were developed 1-2 months later).

In situ hybridization was then used to confirm elf expression in 11.5 heart and liver as well as to determine its expression pattern during earlier liver development, as will be set forth below in the Examples. The liver bud, which originates from foregut endodermal cells, grows into the septum transversum at the 9th day of gestation (13-20 somite stage). Between days 10.5 to 11.0 post coitus, a considerable degree of differentiation occurs: The liver enlarges substantially over this period, this increase in volume being due to the invasion of the mesenchyme of the septum transversum by the hepatic cords, and the initiation of hematopoietic activity in the liver. At day 9.5, a strong labeling of elf becomes apparent in the heart, and the pattern appears to be trabecular, including the wall of the cardiac anlage. A section of the sino-atrial chamber wall also shows a high intensity of elf expression. The surrounding tissue, particularly the caudal liver bud region does not show the presence of silver grains.

At the next stage, day 10.5, silver grains clearly highlight the developing liver, which appears as a horizontal structure (L) in this section. At this stage, the signalling is weakening in the developing heart tissue. The surrounding tissues are remarkable for the absence of silver grains. At day 11.5, a strong labeling of elf becomes apparent in the liver, which is larger in size. The heart at this stage only shows a weak signal posteriorly. As a control, in addition to sense probes, a riboprobe to alpha fetoprotein outlines the developing embryonic liver at days 11-12.

A comparison of the day 9.5 and 10.5 embryos, demonstrates a temporal and spatial expression of elf: the temporal gradient of a rise and fall of elf expression in the heart can be inferred from the strong staining in the developing heart at day 9.5 followed by a weaker staining at the next stage (day 10.5). Simultaneously, liver expression increases. The spatial gradient is apparent where silver grains increase in density on moving from the developing heart to the liver: at day 10.5, antisense RNA probes from elf cDNA hybridized specifically to day 9.5 cardiac mesenchymal tissue; expression at day 10.5 being restricted to cardiac and hepatic tissue, with elf expression finally being restricted to the liver in later 11.5 day embryos. Of note, elf expression was seen in embryonic livers at later stages (days 12.5, 14.5 p.c.), but only in decreasing abundance: the message being detected in these later stages when Northerns and in situs were developed a considerable time later. Sense probes to elf did not hybridize to any tissues. This indicates that ELF expression is not a sudden “on” “off” phenomenon, but more of a gradient pattern: consistent with the expression pattern of brain beta spectrin.

Alpha fetoprotein antisense RNA probes hybridized specifically to 11.5, 12.5, 14.5 embryonic mouse liver tissue, which is in agreement with previous studies of mRNA isolated from embryonic liver samples. The earliest stage that we were able to detect alpha-fetoprotein mRNA by in situ hybridization was at 10.5-11.0 days of gestation. Similar experiments with albumin mRNA have shown it to be expressed at day 9.5 in clusters of cells arising from foregut epithelium and in cords of cells seen to be invading the septum transversum. In experiments with alpha-fetoprotein, the liver was labeled at all subsequent stages (day 11 onwards), and, upon histological examination appeared to occur primarily in the endothelial cells. Hematopoietic cells appeared retractile but did not contain the hybridization grains that were visible over the alpha-fetoprotein positive cells. These experiments show that elf mRNA is localized to early embryonic heart, and then moving to ell liver.

Next, it was determined that elf was a marker for the mesodermal component of liver formation. As Northern analysis had revealed elf expression to occur in day 11.5 heart and liver tissue, in situ localization was performed to investigate whether elf expression was restricted specifically to mesodermal tissue from the heart and the liver and was then compared to the endothelial expression of alpha fetoprotein. The main regions of mesoderm in the developing embryo are dorsal (somitic), intermediate, and lateral. Specifically, lateral plate mesoderm comprises somatic tissues (pleura, pericardium, peritoneum and limb bud), and splanchnic tissues (heart, epicardium, myocardium, connective tissue and smooth muscles of viscera and blood vessels, hemangioblastic tissue, adrenal cortex and spleen). The developing heart, at day 9 (13-20 somites), appears to be only region within the embryo where the endothelial elements of the circulation are surrounded by a vessel wall. The walls of the common ventricular and atrial chambers show an increasing degree of trabeculation. The space between the endothelial and myocardial elements is filled with loose mesenchyme called cardiac jelly. In situ hybridization of days 9 and 10 embryonic heart tissue using elf antisense riboprobes showed high levels of labeling to both the atrial and ventricular regions, highlighting the trabeculation.

Hepatic mesenchyme also originates from lateral plate mesoderm. The septum transversum part of the hepatic mesenchyme originates from the splanchnic mesoderm of the precardiac area and this is considered to be responsible for the subsequent differentiation of hepatocytes. However, tissue explant experiments have demonstrated that all derivatives of the lateral plate can replace hepatic mesenchyme for these later events. The initial experiments have shown that migrating endoderm must interact with mesenchyme for the former to differentiate into hepatocytes and recent studies investigating albumin mRNA expression, an indicator of hepatocyte differentiation, have confirmed these features; Initial expression of albumin mRNA occurs during the invasion of the septum transversum, when foregut endodermal cells clearly contact cardiac mesenchymal tissue. Similarly, primer extension analysis of albumin transcription has shown that the start site of transcription to occur at day 10.5 with a 15-20 fold increase in albumin mRNA upon liver organ formation by day 12.5. In our experiments using alpha fetoprotein as a marker for differentiated hepatocytes, it was obvious that while alpha fetoprotein expression is restricted to the later endodermal component of liver development, elf expression seems to occur in the loosely organized, lighter staining mesenchymal cells, initially cardiac mesenchyme (at day 9.5), then in both cardiac and hepatic tissue (at day 10.5) and then restricted to liver tissue (day 11.5 onwards); elf expression then decreases in abundance upon full embryonic liver formation. Examination of later histological sections (days 11 onwards) demonstrated a diffuse distribution of grains, and the hybridization signal with elf appeared to be localized in the perisinusoidal cells, but not in the hepatocytes.

That elf is expressed in early cardiac mesoderm, with subsequent expression being limited to hepatic mesoderm, indicates that this is a novel marker for the mesodermal component of liver development. Molecular markers have been invaluable in the dissection of inductive events in embryological studies. For instance, in Xenopus, vg-1, a member of the TGF-Beta family, now considered to be the strongest candidate for dorsal mesoderm induction, was in fact originally isolated by differential screening of mRNAs localized in the vegetal hemisphere of developing Xenopus eggs. Activins and other genes belonging to the TGF-Beta family such as vg-1, as well as wnt and BFGF families, represent components of the cascade leading to the commitment to particular mesodermal fate and all are strong candidates as mesoderm-inducing factors. Yet of these, only vg-1 has been demonstrated to be localized to the vegetal cells, the blastomeres responsible for mesoderm induction in vivo. Specific localization of vg-1 was vital and responsible for the persistence required in investigating its role as the inductive agent in mesoderm formation. Similarly, in isolating putative inductive agents required for liver formation, a key step is the localization of a new mRNA isolated from the embryonic livers. Accordingly, it is contemplated that elf and its associated regulatory genes will be of enormous potential benefit as a liver growth factor.

Further characterization of elf.has involved RNA analysis of adult mouse and human tissues, and it was determined that elf hybridizes to adult liver, kidney and testis as a 2.4 Kb transcript in liver and kidney and a 2.6 Kb transcript in adult testis, in very low abundance: both blots were developed after being exposed to film for over a month at −70° C. Genomic DNA analysis of elf expression in DNA (genomic) from human, monkey, rat, mouse, dog, cow, rabbit, chicken and yeast indicates that elf is conserved across the species, being represented in all except rabbit DNA.

In vitro transcription and translation of elf, the latter using nuclease-treated rabbit reticulocyte lysate (promega) has revealed a 34 Kd protein, which is as predicted by the elf insert size and indicating that this insert is in frame for the coding sequence for a specific protein. These studies have established the principle that specific mesodermal mRNAs are localized in a way that guarantees their subsequent segregation to specific mesodermal tissue, in this case the presumed mesodermal component of the liver as shown by embryonic explant studies.

The elf protein has been sequenced, and it has been determined that at least three specific elf protein genes can be identified during early liver development. The sequences for these genes, known as elf-1, elf-2, and elf-3, are shown in the FIGS. 2A-2E, 2F-2I and 2J, respectively. As indicated above, it appears that the elf proteins 1-3 are probably important for the formation of the biliary tree during early liver development. Accordingly, it is contemplated that in accordance with the present invention, the elf proteins will be useful in treating various disorders associated with liver function, including cholestasis, biliary stones, obstruction, stricture, primary biliary cirrhosis, and primary sclerosing cholangitis. As would be readily apparent to one skilled in the art, methods of treatment using the elf proteins would comprise administration of an amount of an isolated elf protein that is effective to treat the specific disease condition described above. As also would be apparent, the elf proteins themselves can be prepared in a number of suitable ways by expression from the nucleic acid sequences indicated at FIGS. 2A-2J including recombinant methods of producing these proteins, followed by separation, isolation and/or substantially purifying the elf proteins. The elf proteins once obtained in this manner can be put into any suitable form that is acceptable for use with patients. In addition, any of these three elf proteins may be administered with any other suitable compound normally utilized for administration into a patient, such as a suitable pharmaceutically acceptable carrier.

Another protein that has been identified and isolated in accordance with the present invention and which is contemplated to be used in a variety of therapeutic methods is known as praja-1. Praja-1 has now been studied in conjunction with the examination of early developing liver proteins, and an analysis of the amino acid translation revealed the presence of a COOH-terminal RING-H2 motif, which is a zinc finger variant. Additionally, Northern blot analysis of RNA from adult mouse showed expression of 3.1, 2.6, and 2.1 kb transcripts in liver, brain, and kidney, and an additional 2.3 kb transcript in testis. Expression of praja-I is also apparent in a colon cancer cell line, SW 480, and as set forth below, it is also contemplated that the praja-1 protein will be a useful marker in early detection of colon cancer.

It has also been learned that praja-1 maps to chromosome X, at about the 36 cM position. Other genes mapping to this general region include moesin (Msn), androgen receptor (Ar), interleukin-2 receptor gamma (IL-2rg), X-linked zinc finger protein (Zfx), and tabby (Ta). The syntony and conserved gene order between mouse and human X chromosomes allows comparison with human disease genes in the region. Human diseases in this region with mesodermal involvement include anhidrotic ectoderm dysplasia (eda) and sideroblastic anemia with spinocerebellar ataxia (asat), and it is thus contemplated that in accordance with the present invention, praja-1 will be useful in treating these disease conditions, as well as degenerative neurological disorders.

In vitro expression of praja-1 has shown that the translational product, which ran as two closely spaced bands of Mr=55.6 and 56.9 kD, is larger than the predicted ORF size of 47.4 kD. One possible explanation is that the expression product is very acidic, and acidic proteins such as granins are known to give anomalously high Mr on SDS-PAGE. The presence of two products suggests translation initiation at a second, internal ATG codon, such as at Met-19.

In addition, antisense studies to praja-1 demonstrated that praja-I is essential for liver architecture formation. Preliminary antisense studies were performed at 1.25, 2.5 and 5 mfn concentrations, utilizing two different ODNs to praja-1. In these tests, liver and block explants were treated with these antisense ODNs compared with control (scrambled, sense or no ODNS). The results showed that control livers were generally larger than the antisense-treated livers, and control blocks showed early hepatocyte growth, cartilage growth, and very preserved bile ducts. Both livers and blocks treated with either antisense ODN to praja-1, showed minimal hepatocyte growth, cell necrosis, yet preservation of cartilaginous tissue, in a dose dependent manner.

In praja-1, aside from the RING-H2 finger, the stretch of thirty-four COOH-terminal amino acids just past this motif is especially rich in proline residues (17.6%); and, as stated, the protein in general is very acidic. Proline-rich domains are found in several mammalian transcription factors, such as that at the COOH-terminus of transcription factor CTF. Proline-rich regions and also acidic regions are likely to function in contacting other proteins. When considering the praja-1 sequence as a whole, the rat Neurodapl gene has the highest similarity. Neurodap1 is expressed abundantly in rat brain, with much smaller amounts in heart and skeletal muscle. Though praja-I likewise shows expression in brain, unlike Neurodap1 (which is a larger 4.8 Kb transcript), it also expresses in liver and kidney. The subcellular localization of Neurodap1 was shown to be concentrated around the endoplasmic reticulum (ER) and golgi of the cerebral cortex and facial nucleus, and especially in the postsynaptic density region of axosomatic synapses. Based on its subcellular localization, plus the presence of the RING-H2 finger, Neurodap I is probably linked to the secretory or protein sorting. This similarity to Neurodap1 indicates that praja-I is most likely involved in protein-protein interactions, possibly in a protein sorting or secretary pathway involved during hepatocyte formation.

The gene coding the praja-1 protein has been sequenced, and this nucleic acid sequence is shown in FIGS. 3A-3B. As indicated above, it appears that the praja-1 protein is probably important for iron transport, and essential for hepatocyte formation as well as hematopoiesis. Accordingly, in accordance with the present invention, praja-1 can be used in methods of diagnosing and treating diseases such as end stage liver disease, iron storage disorders, hepatocellular carcinoma, as well as anemia, such as sideroblastic anemia, ataxia, such as spinocerebellar ataxia, and hemochromatosis. As would be recognized by one skilled in the art, these methods of treatment would involve administering of an effective amount of the praja-1 protein to the patient afflicted with one of the disease conditions set forth above. In addition, the isolation of the praja-1 protein could be obtained by expression of the nucleic acid sequence indicated at FIGS. 3A-3B which codes for the praja-1 protein, and this protein can be produced from its nucleic acid sequence in any suitable manner well known in the art such as recombinant means. Once isolated in this manner, the praja-1 protein can be obtained in a desired form, such as in substantially purified condition, and can be incorporated into any suitable mode of treatment that would be compatible with the patient in need of such treatment. In addition, the praja-1 protein may be administered with any other suitable compound normally utilized for administration into a patient, such as a suitable pharmaceutically acceptable carrier.

Even further, as indicated above, it has also been discovered that the protein praja-1 has been identified in cancerous colon tissue, such as in colon cancer cell line SW 480, which normally does not produce this protein. Accordingly, it is contemplated that in accordance with the present invention, a method of detecting and diagnosing colon cancer is provided wherein colon cells or tissues are taken from a patient being tested, and these cells or tissues are screened in any suitable manner which would identify the presence or absence of the praja-1 protein in the tested cells or tissues. In this manner, the identification of praja-1 in the colon cells or tissues from the patient will be indicative of a cancerous condition in the colon cells or tissues, and thus the present invention will provide a simple and effective method for determining at an early stage, when the disease is still in a treatable condition, if the patient appears to have contracted colon cancer. Conversely, the absence of praja-1 will generally be indicative of a non-cancerous state in tested colon cells.

Still other genes coding for early developing liver proteins in accordance with the present invention have been identified and sequenced, and these proteins will also be useful in various methods of diagnosis and treatment of disease conditions associated with the liver or liver function. Included in these additional genes are those nucleic acids coding for a protein identified as pk, as depicted in FIGS. 4A-4B, nucleic acids coding for a protein identified as protein 106, as shown in FIG. 5, and genes 20, 36, 41, 112, 114, 118 and 129, as shown in FIGS. 6-12. These proteins also appear to useful in hepatocyte formation and in treating liver diseases in a similar manner to many of the proteins discussed above, and in a manner similar to known growth factors should be useful in treating a variety of conditions. For example, protein pk appears to be important in Ito cell formation and fibrosis and thus appears to be useful in the same manner as protein liyor-1 (145). Accordingly, the protein pk, as prepared from the nucleic acid sequence indicated at FIG. 4, will likely be useful in treating end-stage liver disease, hepatocellular carcinoma, as well as other disease conditions including anemia, ataxia, and hemochromatosis. As in the above cases, these early developing liver proteins may be administered with any other suitable compound normally used for administration to patients, such as suitable pharmaceutically acceptable carriers.

Another aspect of the present invention will comprise raising antibodies to the early developing proteins identified above, or to peptides or fusion proteins such as the pk protein (also known as itih-4) derived from these proteins. As would be recognized by one of ordinary skill in the art, antibodies to these proteins or to selected peptides or fusion proteins derived from these proteins may be prepared in any suitable conventional manner currently known, including raising antibodies in such animals as rabbits, sheep, goats, or guinea pigs. In the preferred embodiment, the following antibodies have been raised in rabbits:

(1) Peptides (13-mer) at aa 2-14 of mouse elf gene N-terminus having the sequence 5-ELQRTSSVSGPLS-3.

(2) Peptides (14-mer) at aa 2140-2154 of mouse elf gene C-terminus having the sequence 5-FNSRRTASDHSWSG-3

(3) Peptides (13-mer) at aa 144-156 of mouse prajal gene middle portion having the sequence 5-LRRKYRSREQPQS-3.

In addition, the invention also comprises antibodies to the following peptides:

(1) 145peptide-A (18-mer) which was designed from the C-terminus of gene 145 (Cded) and has the sequence 5-SAQSLVVTLGRVEGGIRV-3 OR 5-CSAQSLVVTLGRVEGGIRV-3.

(2) 145peptide-B (17-mer) which was designed from the middle part of gene 145 (Cded) and has the sequence 5-KIEGSSKCAPLRPASRL-3 or 5-CAPLRPASRLPASQTLG-3.

(3) g59peptide-A (16-mer) which was designed from the N-terminus of gene G59 (Praja1) and has the sequence 5-PPREYRASGSRRGMAY-3 or 5-PPREYRASGSRRGMAYC-3; and

(4) g59peptide-B (15-mer) which was designed from the middle part of gene 59 (Praja1) and which has the sequence 5-CKVPRRRRTMADPDFW-3.

The invention also comprises antibodies to a fusion protein such as a 40 kD pk/itih-4 fusion protein which covers the two EF-hands motifs of itih-4 (about 400-bp 14kD).

The invention further comprises the use of the elf proteins of the present invention in interactions with TGF-β signaling molecules such as Smad2 and Smad3 so as to prevent or treat liver diseases such as primary biliary cirrhosis (PBC) and other diseases involving bile ducts. Evidence has shown that SMAD2 and SMAD3 insufficiency leads to a loss of bile ducts, and thus that TGFβ treatment of normal livers results in an increase in bile duct formation via the activation of SMAD2 and SMAD3. SMAD2/3 activity may be mediated by ELF, a Beta Spectrin. Loss of ELF function results in T lymphocytic proliferation and absent intrahepatic bile ducts. Livers deficient in SMAD2 and SMAD3 exhibit perturbations in ELF localization. This phenotype is seen in Primary Biliary Cirrhosis (PBC), a cholestatic disease with a progressive loss of intrahepatic bile ducts. Perturbations in ELF are correlated with a lack of SMAD2 and SMAD3 in this disease. Immunoprecipitation studies show that ELF binds SMAD2 and SMAD3, and that this binding is increased in PBC.

Previously, it was observed that compound haplo-insufficiency at the smad2 and smad3 loci resulted in a failure to form intrahepatic bile ducts, and that HGF could rescue this phenotype in a SMAD-independent pathway. It was noted that TGFβ could rescue the bile duct insufficiency in the mutant livers, although it did not completely rescue the hepatocytic defects seen. The effect of TGFβ on the wild-type explants was also quite interesting. Treatment of fetal livers with exogenous TGFβ in vitro resulted in a marked increase in the number of intrahepatic bile ducts. Moreover, the morphology of the bile ducts formed underwent a dramatic change. The bile ducts increased to twice their normal size, and were less regularly organized than those found in untreated liver explants.

In addition, it has also been previously shown that HGF was able to direct the formation of bile ducts while bypassing SMAD activation. The question of whether SMAD2 and SMAD3, the pathway specific SMAD proteins downstream of TGFβ and activins, were activated in the livers in the presence of TGFβ was also examined by looking at the subcellular localization of these SMAD proteins by immunoflourescence and confocal microscopy in explant livers from smad2+/−; smad3+/− mutants cultured in the presence of TGF Beta. It was determined that a narrow expression domain of SMAD2 is found just adjacent to the developing bile ducts, in which some SMAD2 appears to be nuclear, suggesting that it is activated and is transducing the TGFβ signal. SMAD3 was also expressed adjacent to the forming bile ducts, although its expression domain was much wider that seen for SMAD2. SMAD3 could also be found in the nuclei of some cells, suggesting it too was activated in response to the TGFβ. Therefore, diminution of SMAD activity through genetic haploinsufficiency ablates bile duct formation, while exogenous activation of SMAD2 and SMAD3 can augment bile duct development, suggesting a central role of TGFβ and SMAD2 and 3 in bile duct formation.

The phenotype of the smad2+/−; smad3+/− embryos was reminiscent of what was seen when ELF3 was inhibited by antisense oligonucleotides in liver explants (Mishra, Oncogene, 1999). Specifically, bile ducts failed to develop, and the hepatocytic architecture was highly deranged. ELF3 is a β-spectrin protein in accordance with the present invention, which we have shown is expressed in the membrane of hepatocytes. Indeed, recent confocal experiments have pinpointed a focused concentration of ELF3 protein at the apical, canalicular surface of the hepatocytes, which led to an examination of the effect of smad2/3 ablation on ELF localization. The evidence regarding the association of smad2 and 3 and the ELF proteins of the present invention is discussed in more detail in Example 3.

It is thus submitted that the foregoing embodiments are only illustrative of the claimed invention, and alternative embodiments well known or obvious to one skilled in the art not specifically set forth above also fall within the claimed scope.

In addition, the following examples are presented as illustrative of the claimed invention, and are not deemed to be limiting of the scope of the invention, as defined by the claims appended hereto, in any manner.

EXAMPLE 1

In accordance with the cloning strategy of the present invention to identify genes involved in early mouse liver development, we have isolated Praja-1, a gene with similar sequences to the Drosophila melanogaster gene goliath (gl), and which is involved in the fate of mesodermal cells ultimately forming gut musculatures, fat body, and the heart. Praja-1 is a 2.1 kb gene encoding a putative 423 amino acid ORF and includes a COOH-terminal RING-H2 domain. Using the Jackson Laboratory BSS panel, we have localized praja-1 on chromosome X at 36 cM, near the X inactivation center gene, Xist. Northern blot analysis demonstrated three transcripts (3.1, 2.6 and 2.1 kb) in mRNA from adult mouse tissues brain, liver, and kidney as well as in mRNA from developing mouse embryos (days 7, 11, 15 and 17 post coitus, or p.c.). In vitro transcription/translation yielded two products with a Mr of 55.6 and 56.9 kD. The presence of the RING-H2 domain, a proline-rich region at the COOH-end, and regions rich in acidic amino acids, leads to the hypothesis that the Praja-1 product is involved in mediating protein-protein interactions, possibly as part of a protein sorting or transport pathway. This is strengthened by the similarity of praja-1 to rat Neurodap1, whose product has been shown to localize to the endoplasmic reticulum and golgi in brain.

The molecular mechanisms underlying hepatocyte differentiation are not well understood, and thus identifying the genes underlying the control of liver development will provide powerful tools for understanding liver function and development, and will allow the use of inducing liver differentiation for therapeutic purposes. As part of a strategy to clone such genes, we isolated a new RING-H2 finger gene, praja-1. RING-H2 fingers, a type of zinc finger, are similar to RING fingers except that Cys4 is replaced by His (see Freemont, Ann. N.Y. Acad. Sci. 684:174-192 (1993); Lovering et al., P.N.A.S. 90:2112-2116 (1993)). Here we show that praja-1 possesses a RING-H2 motif near the COOH terminal.

The RING-H2 motif is similar to that of the Drosophila melanogaster gl gene (Bouchard et al., Gene 125:205-209, 1993), and to the rat Neurodap1 gene (Nakayama et al., J. Neurosci. 15:5238-5248, 1995). Praja-1, which localizes to chromosome X, is expressed in mouse brain, liver, and kidney. The presence of the RING-H2 motif, plus the acidic, hydrophilic nature of the translation product, leads to the hypothesis that praja-1 plays a role in protein transport.

Materials and Methods

cDNA Preparation and 3′-RACE PCR:

RNA was isolated from livers of day 11 p.c. embryonic mice (ICR, Harlan Sprague-Dawley) using guanidine thiocyanate (Chomczynski et al., Ann. Biochem. 162:156-159, 1987). Poly(A)+ mRNA was isolated from total RNA using Dynabeads, as per manufacturer's instructions. First strand cDNA was made from poly(A)+ mRNA using the Promega Reverse Transcriptase System and the 3′-RACE primer 5′-GACTCGAGTCGACATCGA-T17 (Frohman, In: M. A. Innis et al. (eds.), PCR protocols: a guide to methods and applications, Academic Press, San Diego, pp. 28-38., 1990). The 3′-RACE primer was also used as the reverse primer in the PCR reaction. The forward PCR primer, originally designed to amplify a conserved region of a clone 145/PH (pleckstrin homology) domain, was 5′-CTCAAGCAGGTCCTGGCACA. The PCR reaction mix contained cDNA from about 10 ng of poly(A)+ mRNA, 25 pmol of each primer, 1 mM dNTP mix, and 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer) all in 10 mM Tris, 1.5 mM MgCl₂, and 75 mM KCl, pH 9.2 in a final volume of 50 ml. The temperature program comprised 35 cycles of denaturation (94° C., 1 min), annealing (55° C., 1 min), and extension (72° C., 3 min), followed by an additional 8 minute extension. One of the resulting PCR products (CH7) comprised a 725 bp fragment, which was cloned into vector PCRII using the Invitrogen TA Cloning Kit for sequencing, and found by sequence analysis to possess a RING-H2 finger. The portion of the final cDNA clones which correspond to CH7 is indicated in FIG. 3.

Library Screening:

The PCR product CH7 was labeled with [a-32P]-dCTP (3000 Ci/mmol, Amersham) via primer extension using the reverse PCR primer plus AmpliTaq polymerase at 72° C. in PCR buffer (Konat et al., in PCR Technology: Current Innovations (H. G. Griffin and A. M. Griffin, Eds.), CRC Press, Boca Raton, pp. 37-42, 1994). The resulting antisense probe was used to screen plaque lifts of a whole embryonic mouse (day 11 p.c.) cDNA library in vector λZap (Stratagene). Positive plaques were picked and purified, and DNA was isolated from lysates using standard procedures (Silhavy et al., Experiments with gene fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1984). Inserts were excised from λZap DNA using EcoRI, and were subcloned into pGEM3Zf(−) (Promega) for sequencing and subsequent manipulations.

DNA Sequence Analysis:

DNA sequence comparisons to existing sequences were performed utilizing BLAST searches in Genbank. Alignments were performed using the GCG program PILEUP.

Chromosomal Mapping:

Southern blot analysis of genomic DNA from C57BL/6J (B6) and Mus spretus (SPRET/Ei) using [32P]-labeled CH7 as a probe revealed a restriction fragment length polymorphism for the enzyme TaqI. This polymorphism was used to follow the inheritance of the praja-1 gene using the (B6 X SPRET/Ei) X SPRET/Ei backcross panels (BSS) from The Jackson Laboratory Backcross DNA Panel Map Service (Rowe et al., Mammalian Genome 5:253-274, 1994). Linkage and order relative to other markers was determined by minimizing the number of multiple recombinants within each haplotype.

Northern Blot Analysis of Praja-1 Expression:

Northern blots containing 2 micrograms of poly(A)+ mRNA from mouse tissues (Clontech) were probed with [32P]labeled CH7 antisense strand using Express Hyb hybridization solution (Clontech) at 68° C., washed according to manufacturer's instructions, and subjected to autoradiography. A [32P]-labeled b-actin probe supplied with the Northern blots was used as a control to normalize RNA levels in each lane.

In Vitro Transcription/translation:

A transcription/translation-coupled rabbit reticulocyte lysate system (Promega) was used, as per manufacturer's instructions for [35S]methionine labeling. Clones of praja-1 in pGEM3Zf(−) plus a luciferase control clone were used with T7-RNA polymerase (sense direction). Each reaction comprised 12.5 ml rabbit reticulocyte lysate, 1 ml reaction buffer, 0.5 ml 1 mM amino acid mix minus methionine, 0.5 ml T7-RNA polymerase, and 20 units RNasin, all in 25 ml final volume. After a 90 min incubation at 300° C., products were lysed in SDS/mercaptoethanol treatment buffer and separated on a 10% SDS-polyacrylamide gel according to Laemmli, Nature 227:680-685 (1970). Proteins were electroblotted onto a BAS-NC membrane (Schleicher & Schuell) using a BioRad Trans-Blot apparatus according to manufacturer's instructions. Labeled products were visualized by autoradiography.

Results:

Isolation and Sequence Analysis of the Novel Gene, Praja-1:

As part of the analysis of genes involved in liver development and function, we amplified the 3′ end of a previously undescribed gene, CH7. We used the CH7 probe to screen a mouse embryonic cDNA library and isolated two overlapping clones, praja-1-5 and praja-1-6. Sequence analysis of the consensus overlap region revealed an open reading frame (ORF) of 424 amino acids, with a predicted size of 47.4 kD. Hydropathy analysis (Kyte et al. J. Mol. Biol. 157:105-132, 1982; not shown) shows that the translation product is highly hydrophilic, with no hydrophobic leader or membrane-spanning regions. The translation is also very acidic, with a pI of 4.6 and containing 17.7% acidic residues (Asp plus Glu). The putative ATG start codon indicated in FIG. 3 was selected because it is the upstream-most ATG that is in-frame with the ORF, and is preceded 21 bp upstream by a TAG stop codon. The context of this ATG, however, is only a weak fit to the consensus Kozak recognition sequence GCCACCatgG in that it does not have a purine at −3 nor a G at +4 (reviewed by Kozak, Genome 7:563-574, 1996). Sequence analysis of the amino acid translation revealed the presence of a COOH-terminal RING-H2 motif, which is a zinc finger variant (Freemont, supra). FIG. 13 shows an alignment of the RING-H2 motif of praja-1 with those of several other RING-H2 containing proteins.

Linkage Analysis Places Praja-1 on Mouse Chromosome X:

A restriction fragment length polymorphism for praja-1 was identified using CH7 as a probe on a Southern blot containing DNA from the two parental strains digested with several restriction enzymes (TaqI, BglII, EcoRI, EcoRV, HindIII, HincII, KpnI, PstI). For every enzyme used, C57B16/J had only a single restriction fragment, while two fragments were always observed within the SPRET/Ei lane. A polymorphism obtained using TaqI was used to type the inheritance of the C57B1/6J allele in the BSS panel. There are two Spretus bands S1 and S2 and one C57B1/6J band B1. After comparison of the praja-1 genotypes to other genes typed within the database, it was determined that praja-1 maps to mouse chromosome X at about the 36 cM position (FIG. 14). The S1 band is the praja-1 allele on X chromosome of SPRET/Ei. The S2 TaqI fragment appears in every backcross animal. Since all males from the backcross contain this allele, it is not localized to the X chromosome. Since females also have the S2 band, it is not Y-linked. Therefore S2 is an autosomal locus that contains sequence homology to the praja-1 probe sequence. Other genes mapping to this general region include moesin (Msn), androgen receptor (Ar), interleukin-2 receptor gamma (Il2rg), X-linked zinc finger protein (Zfx), and tabby (Ta). This area is also 1.1 +/−1.1 cM from the Xist locus. Further studies are needed to determine if praja-1 is not expressed on inactivated X-chromosomes and if it plays a role in X-inactivation. The syntony and conserved gene order between mouse and human X chromosomes (Herman et al., Genome 6:S317-S330, 1996) allows comparison with human disease genes in the region. Human diseases in this region with mesodermal involvement include anhidrotic ectoderm dysplasia (eda) and sideroblastic anemia with spinocerebellar ataxia (asat).

In vitro expression produces a protein product larger than the predicted size. An autoradiogram of the in vitro transcription/translation products of clones praja-1-5 and praja-1-6 showed that only praja-1-5 produced a significant product. The product, which ran as two closely spaced bands of Mr=55.6 and 56.9 kD, is larger than the predicted ORF size of 47.4 kD. One possible explanation is that the expression product is very acidic, and acidic proteins such as granins are known to give anomalously high Mr on SDS-PAGE (Huttner et al., Trends Biol. Sci. 16:27-30, 1991). The presence of two products suggests translation initiation at a second, internal ATG codon, such as at Met-19 (FIG. 3).

Praja-1 transcripts are present in embryonic and in mouse tissues. Northern blot analysis of RNA from adult mouse showed expression of 3.1, 2.6, and 2.1 kb transcripts in liver, brain, and kidney, and an additional 2.3 kb transcript in testis. The praja-1 protein is unlikely to be a membrane receptor, since it lacks a hydrophobic transmembrane domain. The uniform hydrophilicity suggests a soluble protein. The praja-1 RING-H2 motif is shown aligned with those from several other proteins in FIG. 13. RING fingers are generally thought to function in protein-protein interactions (Borden et al., Curr. Opinion Struct. Biol. 6:395-401, 1996; Saurin et al., Trends Biochem. Sci. 96:208-214, 1996). To cite a specific example, if either of the two cysteines that comprise the Zn++ binding site of the RING finger of acute promyelocytic leukemia protooncoprotein PML are mutagenized, then the nuclear multiprotein complex, or so-called nuclear bodies, fail to occur (Borden et al., EMBO J. 14:1532-1541, 1995). The authors conclude that the PNM RING domain, and probably other RING finger domains, are involved in protein-protein interactions.

In praja-1, aside from the RING-H2 finger, the stretch of thirty-four COOH-terminal amino acids just past this motif (FIG. 3) is especially rich in proline residues (17.6%); and, as stated, the protein in general is very acidic. Proline-rich domains are found in several mammalian transcription factors, such as that at the COOH-terminus of transcription factor CTF, and proline-rich regions and also acidic regions are likely to function in contacting other proteins (Mitchell et al., Science 245:371-378, 1989). A BLAST search of the proline-rich COOH-terminus revealed no significant matches to any protein in the available databases, however, when considering the praja-1 sequence as a whole, the rat Neurodap1 gene has the highest similarity; the alignment is presented in FIG. 15.

Neurodap1 is expressed abundantly in rat brain, with much smaller amounts in heart and skeletal muscle. Though praja-1 likewise shows greatest expression in brain, unlike Neurodap1 it also expresses in liver and kidney. The subcellular localization of Neurodap1 was shown to be concentrated around the endoplasmic reticulum (ER) and golgi of the cerebral cortex and facial nucleus, and especially in the postsynaptic density region of axosomatic synapses (Nakayama et al., supra). Based on its subcellular localization, plus the presence of the RING-H2 finger, the authors concluded that Neurodap1 is probably linked to the secretary or protein sorting. Praja-1 does differ from Neurodap1 in several respects, however. In addition to being expressed in some different tissues than Neurodap1, praja-1 encodes for a product that is smaller (47.4 kD, based on the composite of the clones in FIG. 3) vs. 77.9 kD for Neurodap1. The difference in size is at the N-terminus of the proteins (FIG. 15). The largest transcript we observed for praja-1 was 3.1 kb, whereas Neurodap1 exists as a single 4.8 kb transcript on Northern blots of rat brain mRNA.

In light of the fact that BRCA1, which possesses a RING finger, has an acidic pI, and is a secretary protein, also has properties of the granin family of proteins (Jensen et al., Nature Genet. 12:303-308, 1996), we examined praja-1 for a granin signature. We found no region in the praja-1 translation that gave a perfect match to the consensus E[N/S]LX[A/D]X[D/E]XEL, though two regions matched five of the seven conserved residues. We were also unable to demonstrate the presence of clear coiled-coils, which are present in BRCA1 and proteins with the previously-mentioned tripartite structures. In these respects, praja-1 is more similar to Neurodap1 than to proteins such as BRCA1. Also, though the RING-H2 finger in praja-1 shows much similarity to that from the D. melanogaster goliath (gl) protein (FIG. 13), the goliath protein possesses an alkaline pI (8.9) and no sequence similarity to praja-1 outside of the RING-H2 finger. The RING-H2 motif plus acidic and proline-rich regions, and similarity to Neurodap1, leads to the conclusion that praja-1 is involved in protein-protein interactions, possibly in a protein sorting or secretory pathway.

EXAMPLE 2

In accordance with the present invention, investigations were made with regard to the induction of differentiation in liver tissues in order to isolate and identify early developing liver proteins for use in therapies involving the liver and liver functions. In the developing fetus, inductive interactions, intercellular communication and the establishment of cell polarity are critical for growth and patterning during development. However, the precise mechanisms by which these effect hepatocyte differentiation or liver development have not previously been elucidated. Mammalian liver development was first recognized to be established through a specific sequence of interactions between mesenchymal and endodermal embryonic tissues. At 9.5 days of mouse gestation, upon signaling from the cardiac mesenchyme, endodermal cells from the liver diverticulum proliferate and migrate into the surrounding septum transversum. This specific area of loose mesenchyme in turn differentiates into hepatic mesenchyme and a liver bud is finally recognizable microscopically at about 10.5 days of gestation. This hepatic mesenchyme is continually responsible for the hepatocyte proliferation which then proceeds throughout embryonic life (Le Douarin, Med. Biol. 53:427-455, 1975). Albumin transcription can be detected as early as at day 9.5 (Cascio et al., Development 113:217-225, 1991), implying that hepatocyte differentiation begins when hepatic endoderm comes into contact with cardiac mesoderm. As a first step towards the analyses of signal transduction pathways regulating such a restricted pattern of gene expression, molecular markers as well as regulatory genes are required to identify the interactions required for liver development.

The dissection of gene regulatory pathways in the liver has led to the identification and characterization of transcriptional activators, C/EBP, DBP, LFB 1/HNF 1, 3 and 4 (Johnson, Cell Growth Differ. 1:47-52, 1990; Kuo et al., Development 109:473-481, 1990; Frain et al., Cell 59:145-157, 1989), of liver specific genes, such as α-fetoprotein and albumin (Tilghman, Oxford Surveys on Eukaryotic Genes, Oxford University Press, 1985). Yet, with the exception of HNF4, 3 α and β (Ang et al. Development 119:1301-1315 (1991) and Cell 78:561-574, 1994), none of the above have been found to play a definitive role in determining cell-lineage and regional specification of the developing liver. The small volume of liver buds (approximately 4×10⁻² mm³) yields even smaller quantities of proteins, DNA and messenger RNA thus making the molecular analysis of liver development difficult. Therefore, the construction of early embryonic liver cDNA libraries, and performing subtractive hybridization still remains the most plausible and comprehensive method of obtaining an unbiased catalogue of genes required during early mouse liver development (see Harrison et al, Development 121:2479-2489, 1995).

The isolation of markers would provide further insight into identifying transcriptional activators and growth factors involved in such a restricted pattern of gene expression, and eventually provide an approach to identifying signal transduction pathways involved in hepatocyte differentiation. In some cases, these pathways have been characters as in patterning and axis formation of the vertebrate head and body (Oliver et al., Development 121:693-705, 1995; Kessel et al., Science 249:374-379, 1990). For example, in Xenopus, a network involving brachyury, activin and wnt-related genes, is responsible for mesoderm induction, somitogenesis, myogenic and sclerotomal differentiation (see, e.g., Wilkinson et al., Nature 343:657-659 (1990); Herrmann et al., Development 113:913-917 (1991); Green et al., Trends Genet. 7:245-250 (1991); Sokol et al., Cell 67:741-752 (1991); Smith et al., Cell 67:753-767 (1991), and dorsal ventral axis formation results from Xgsk-3 (the Xenopus homologue of Drosophila zw3/shaggy) phosphorylating its Xenopus homologue of armadillo, β catenin thus regulating the level of β catenin available for dorsal axis formation. However, there are no available molecular markers nor pathways which chamctedw either earlier liver development, nor its crucial mesodermal component.

In accordance with the present invention, it has been possible to identify and characterize such molecular markers and possible inductive transcripts for liver development. As set forth below, the characterization of the elf protein is described, and the expression of this protein may mark the separate components of liver development. The “bottom up” approach with regard to this charatcerization in general has led to the identification of a totally unexpected group of genes, and in particular, this is described with regard to the elf protein, which is probably involved in playing a role in establishing cell polarity by interactions at the surface membrane.

Characterization of cDNA Libraries:

The four stages in liver development (e10, e11, e12, and e14, where e=embryonic) are defined developmental time points from undifferentiated mesodermal/endodermal cells to a well developed and differentiated fetal liver. A change in cell polarity occurs at e9-10. At e10.5-11, invasion and migration of endodermal cells into surrounding mesenchyme occurs; at e11.5-12, pseudolobule formation, cords of hepatocytes form together with early sinusoids. cDNA libraries representing these stages would therefore, represent “captured” mRNA species expressed in greater abundance during crucial time periods for hepatocyte formation, enabling their isolation and providing a method for analyzing the changing pattern of gene expression during liver development.

Libraries containing 5.0×10⁶-4.1×10⁷ independent clones were generated from the largest cDNA fractions. Current estimates demonstrate that a library containing 5.0×10⁵ clones (Sambrook et al, Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989) is a representative library with a 99% probability that rare transcripts (less than ten copies per cell) are present. Our libraries are therefore likely to be truly representative of their respective mRNA species for that stage.

Qualitative and Developmental Profiles of the Libraries:

These were obtained, utilizing genes, such as IGF-II, IGFBP-2, IGF1, C/EBP, HNF/LFBI known to be expressed at different time points in developing liver. The data in Table 2 below demonstrate that IGF-I was not detected in any of the embryonic libraries, while IGF-H was detected in the e10.0 and e12.5 libraries (3 at e10.0 and 4 at e12.5). IGF-II was not detected in the adult liver library. Interestingly, BP-2 clone frequencies are similar to IGF-II in the early e6.5, e7.5 and e8.5 libraries (data not shown), but in the liver cDNA libraries the clone frequencies differed, for BP-2 only one clone per 100,000 being detected at e10.0 and e11.5, while 7 were detected in the adult liver cDNA library compared to the greater numbers for IGF-II. This implied that its temporal and spatial expression in the embryo and fetus is different from IGF-II and this was subsequently confirmed by in situ studies. HNF1/LFB I detected in the e12.5 library was suddenly detected at day 11.5 and 12.5 in low abundance (2 clones/100,000 at e11.5 and 5 at e12.5), confirming that while it is expressed, its level also may be regulated, albeit downward, in embryonic stages. Lastly, mouse β-Actin was used as a reference: all seven libraries had similar β-Actin frequencies from 120-300/100,000 clones which is considered representative of such embryonic libraries.

Identification of Stage Specific Clones by Subtractive Methods:

Two subtracted libraries were then constructed as previously described, comprising 64 clones (e11.5-12.5), and 174 clones (e10.5-11.5). Further characterization of these clones was carried out by Southern hybridization, sequencing, Northern blot analysis, Zoo blot analysis, and in vitro fertilization of protein. Using Southern blotting, thirty-four clones were shown to be stage specific and not containing mitochondrial, ribosomal and globin sequences, and further analysis was carried out on elf.

Identification and Developmental Regulation of Elf Transcripts:

Elf mRNA in tissues from mid-gestational embryos were analyzed, and tissues were dissected from day 11 onwards since it was at this stage that discrete hepatic, cardiac and other tissues could be dissected with ease, and the subsequent RNA isolated was of good quality. Using a 32P-labeled 1.1 Kb insert representing elf, the specificity of the developmental changes in the steady state levels of elf was evaluated by also measuring the relative levels of β-Actin. This revealed a 2.1 Kb transcript at high stringency washes. Scanning densitometry of the respective bands revealed that maximal expression of elf occurred in liver and heart, less so in other tissues but specifically on day 11, and in 12.5, 14.5 in decreasing abundance (when Northerns were developed 1-2 months later).

Sequence Analysis of Elf:

After subtraction hybridization, one stage specific clone was analyzed in detail: sc32. The initial libraries were then screened at high stringency (0.2×SSC, 60°), to obtain overlapping clones for sc32. Positives were picked, and after in vivo excision (Stratagene) into Bluescript, these were sequenced using the dideoxy chain termination method using oligonucleotides corresponding to previously determined sequence. Of the seven clones picked, three were found to be overlapping to sc32 and included sequence encoding elf (FIGS. 16a and 16 b). Confirmation of the identity of the clones and elf was carried out by Northern blot analysis of mouse embryonic tissues. In the case of elf, this gave rise to the same initial 2.1 Kb transcript with sc32 as a probe. A start codon was not present suggesting that we had not cloned the 5′ end of the cDNA. However, the northern blot showed a 2.1 Kb transcript, thus suggesting that we had cloned complete elf and this probably represented a spliced form of β-fodrin. The authenticity of the 3′ end of the elf sequence was confirmed by the comparison of the elf sequence with the expressed sequence tags (EST) database. Although no mouse ESTs for elf sequence were found, three different human EST clones were found to span the region of unique last 100 nt and the 5′ adjacent sequence, suggesting the existence of elf homolog in human cells (see FIGS. 16a and 16 b).

Prior sequence analysis has shown elf to bear 80% identity to β-fodrin, a non erythroid β-spectrin. Our sequence to elf is located between domains II and III of the β-spectrins. Domain II comprises 17 repeats of a 106 amino-acid motif and an ankyrin binding domain (FIG. 16a). The ankyrin binding domain is required for the correct subcellular localization of adducin, ankyrin and the Na+, K+ ATPase, without which cell morphology is disrupted. Domain II comprises a C terminal domain which contains varying numbers of residues (52-265) in alternatively spliced forms giving rise to tissue specific expression (Hu et al., J. Biol. Chem. 267:18715-18722, 1992), as well as the PH domain.

In Situ Localization of Elf:

In situ hybridization confirmed elf expression in 11.5 heart and liver and determined its expression pattern during earlier liver development, using elf sense probes and alpha fetoprotein antisense probes as controls. The hepatic diverticulum, which originates at the foregut-midgut junction, begins to grow into the septum transversum at the 9th day of gestation (13-20 somite stage). Between days 10.5 to 11.0 p.c., a considerable degree of differentiation is seen in this primitive liver. The liver enlarges substantially over this period: the increase in the overall volume being due to the invasion of the mesenchyme of the septum transversum by the hepatic cords, and the initiation of hematopoietic activity in this organ. At day 9.5, a strong labeling of elf becomes apparent in the cardiac silhouette: the pattern appears to be trabecular, including the wall of the cardiac anlage. A section of the cephalad chamber (sino-atrial chamber) wall also bears a high intensity of elf expression. The surrounding tissue, particularly the caudal liver bud region does not show the presence of silver grains. At the next stage, day 10.5, silver grains clearly highlight the developing liver, which appears as a horizontal oriented structure (L) in this section. At this stage, the signaling is weakening in the developing heart tissue. The surrounding tissues are remarkable for the absence of silver grains. At day 11.5, a strong labeling becomes apparent in the liver, which is larger in size. The heart shows an extremely weak signal: silver grains being visible in only a single streak posteriorly. At this stage, elf expression also appears in the umbilical cord. As a control, in addition to sense probes, a riboprobe to α-fetoprotein outlines the developing embryonic liver at day 11-12.

A comparison of the day 9.5 and 10.5 embryos demonstrates a clear temporal and spatial gradient of maximal tissue staining with silver grains representing elf riboprobe: the temporal gradient of a rise and fall of elf expression in the heart may be inferred from the strong staining in the developing heart at day 9.5 followed by a weaker staining at the next stage (day 10.5). Simultaneously, liver expression increases. The spatial gradient is apparent from the developed patterns of thesde tissues which showed that silver grains increase in density as one moves from the developing heart to the liver: at day 10.5, antisense RNA probes from elf cDNA hybridized specifically to 9.5 day cardiac mesenchymal tissue; expression at day 10.5 being restricted to cardiac and hepatic tissue; elf expression finally being restricted to the liver in later 11.5 day embryos. Of note, elf expression was seen in embryonic livers at later stages (days 12.5, 14.5 p.c.), but only in decreasing abundance: message being detected in these later stages when Northerns and in-situs were developed a considerable time later. Elf sense probes did not hybridize to any tissues.

Alpha fetoprotein antisense RNA probes hybridized specifically to 11.5, 12.5, 14.5 embryonic mouse liver tissue, in agreement with previous studies of mRNA isolated from embryonic liver samples (Tilghman et al., P.N.A.S. 79-5254-5257, 1982). The earliest stage of detection of α-fetoprotein mRNA by in situ hybridization was at 10.5-11.0 days of gestation. Similar experiments with albumin mRNA (Cascio et al., Development 113:217-225, 1991) have shown it to be expressed at 9.5d in clusters of cers arising from foregut epithelium and in cords of cells beginning to invade the septum transversum. In the experiments with α-fetoprotein, the liver was labeled at all subsequent stages (day 11 onwards), and, upon histological examination appeared to occur primarily in the endothelial cells. Hematopoietic cells appeared retractile but did not contain the hybridization grains that were visible over the α-fetoprotein positive cells.

ELF mRNA Distribution in Mesodermal Tissues Versus Alpha Fetoprotein mRNA in Endodermal Tissue:

Since Northern analysis revealed elf expression to occur in day 11.5 heart and liver tissue, we investigated whether elf expression was restricted specifically to mesodermal tissue from the heart and the liver and compared this to the endothelial expression of α-fetoprotein. Three main regions of mesoderm can be discriminated in the developing embryo: dorsal (somitic), intermediate, and lateral. Lateral plate mesoderm comprises somatic (pleura, pericardium, peritoneum and limb bud), and splanchnic (heart-epicardium, myocardium, connective tissue and smooth muscles of viscera and blood vessels, hemangioblastic tissue, adrenal cortex and spleen). Regarding the developing heart, at day 9 (13-20 somites), this is seen to beat regularly and strongly. At this stage, the heart appears to be the only region within the embryo where the endothelial elements of the circulation are surrounded by a vessel wall. The walls of the common ventricular chamber as well as the common atrial chamber show an increasing degree of trabeculation. Of note, the space between the endothelial and myocardial elements is filled with loose mesenchyme called cardiac jelly. In situ hybridization of days 9 and 10 embryonic heart tissue using elf antisense riboprobes demonstrated high levels of labeling to both the atrial and ventricular regions.

Hepatic mesenchyme also originates from lateral plate mesoderm. The septum transversum part of the hepatic mesenchyme originates from the splanchnic mesoderm of the precardiac area and this is thought to be responsible for the subsequent differentiation of hepatocytes. However, tissue explant experiments have shown that all derivatives of the lateral plate can replace hepatic mesenchyme for these later events. While these initial experiments have demonstrated migrating endoderm must interact with mesenchyme for the former to differentiate into hepatocytes (Le Douarin, 1975; Houssaint, Cell Differ. 9:269-279, 1980), more recent studies investigating albumin mRNA expression as an indicator of hepatocyte differentiation, have confirmed these features: initial expression of albumin mRNA occurs during the invasion of the septum transversum, when the hepatic precursor cells clearly contact cardiac mesenchymal tissue. Similarly, primer extension analysis of albumin transcription has revealed the start site of transcription to occur at day 10.5 with a 15-20 fold increase in albumin mRNA upon liver organ formation by day 12.5. In our experiments using α-fetoprotein as a marker for differentiated hepatocytes, it was clear under high magnification, that while α-fetoprotein expression is restricted to the later endodermal component of liver development, elf expression seems to occur in the loosely organized, lighter staining mesenchymal cells—initially cardiac mesenchyme (at day 9.5), then in both cardiac and hepatic tissue (at day 10.5) and then restricted to liver tissue (day 11.5 onwards; elf expression then decreasing upon liver formation. Examination of the later histological sections (days 11 onwards) showed a diffuse distribution of grains. The resolution that was attained did not allow one to draw a firm conclusion about the identity of the hybridizing cells, although it seemed that the hybridization signal with elf was localized in the perisunusoidal cells, but not in the hepatocytes.

Distribution of Elf RNA in Adult Tissues, Conservation in Evolution:

Further characterization of elf has involved RNA analysis of adult mouse tissues. Elf hybridizes to adult liver, kidney and testis as a 2.1 Kb transcript in liver and kidney and a 2.6 Kb transcript in adult testis, in very low abundance. Genomic analysis of elf DNA from human, monkey, rat, mouse, dog, cow, rabbit, chicken and yeast indicates that elf is conserved across the species, being represented in all except rabbit DNA (FIG. 18).

In vitro transcription and translation of elf, the latter using nuclease-treated rabbit reticulocyte lysate (promega), has revealed a 34 Kd protein, which is as predicted by the elf insert size and indicating that this insert is in frame for the coding sequence for a specific protein (FIG. 17).

Embryonic Liver Explants Cultures:

One of the goals of the investigations in conjunction with the present invention was to establish a functional assay for determining the developmental roles of elf and ss3 in liver formation. Mouse embryonic liver explants were cultured in our laboratory, in order to overcome the dissection and analysis of extremely small tissue sections at day 10-10.5 when the liver bud is 0.2 mm. When cultured in the complete absence of mesodermal derivatives, hepatic endoderm deteriorates rapidly. Only 2 out of 15 such liver explants survived. Hematoxylin and eosin staining showed a necrotic endoderm with no apparent signs of hepatic differentiation. When associated with the surrounding mesoderm particularly cardiac mesoderm (en bloc dissections), the endodermal cells had proliferated and invaded the mesoderm strands. Hepatocytes were seen to be organized in cords separated by sinusoids with pseudo-lobule formation. All 15 out of 15 cultures from en bloc dissections were completely viable. These studies confirm prior explant studies demonstrating the necessity of surrounding mesoderm for liver formation. Semi-quantitative RT-PCR analyses of elf, other clones ss3, 145, HNF 3β with GAPDH and α-fetoprotein as controls demonstrate increased expression during mesodermal-endodermal interactions.

Early experiments in chick embryos (Douarin, 1975, supra) have demonstrated that at the primitive streak stage, the prospective hepatic area is localized in the middle and in the lateral areas anterior to Hensen's node. At the head process stage, prospective liver areas coincide with cardiac areas, being concentrated in bilateral areas extending from the tip of the head process to an area slightly behind the primitive pit. Potential liver areas were tested by transplantation of pieces of tissue on the chorioallantoic membrane; liver differentiation in such explants was dependent upon the presence of cardiac tissue: no liver tissue was found without cardiac cells in the vicinity, whereas some grafts contained heart tissue without liver. After gastrulation is completed, it is during the somitic stage that the liver and heart segregate partially—the presumptive cardiac mesenchyme migrates anteriorly and venally into the cardiac fold, the prospective myocardial cells becoming incorporated in the heart anlage. Another series of experiments using carbon particle labeling, radiodestruction and coelomic transplantation of pieces of blastoderm showed liver endodermal and mesodermal areas which am superimposed during the early embryonic stages evolve differently later on.

Tissue explant studies have revealed that in normal liver development, hepatocyte differentiation and the formation of liver lobes is entirely dependent upon the mesodermal component which then becomes progressively colonized by the growing endoderm hepatic cords (see Douarin, 1975, supra). These stimulating properties of the cardiac, and then, hepatic mesenchyme have been demonstrated to begin at the 5 somite stage and last throughout embryonic life. The findings set forth herein show that elf is expressed in early cardiac mesoderm, with subsequent expression being limited to hepatic mesoderm, revealing this to be a novel marker for the mesodermal component of liver development. Of note, in normal development, pure liver mesenchyme is never observed. That these explant studies have demonstrated expression of elf, indicates that the elf protein will be useful in identifying and studying such interactions between mesoderm and foregut endoderm.

Summary of Events During Hepatocyte Formation Indicating a Role for Elf:

Embryonic Stage endodermal cell hypertrophy | | | elf day 9.5 change in cell polarity |expression | day 10.5 invasion and migration into surrounding mesenchyme | day 11.5 pseudolobule formation, cords of hepatocytes, early sinusoidal formation | day 14.5 hematopoietic foci and fully differentiated fetal hepatocytes

Sequence analysis has shown elf to bear 80% identity to β-fodrin, a non erythroid β-spectrin. β-spectrins have been implicated in numerous functions including the maintenance of cell surface polarity of cells (Nelson et al., J. Cell. Biol. 108:893-902, 1989); the maintenance of cell—cell junctions (Thomas et al., Development 120:2039-2050, 1994, Luna et al., Science 258:955-964, 1992); β-spectrins contain binding sites for other proteins, such as ankyrin and actin (Hu et al., J. Biol. Chem. 267:18715-18722, 1992; Speicher et al., Nature 311:177-180, 1984). Smaller isoforms β-spectrins have been well described. For instance, a 4.0 Kb muscle tissue transcript is thought to encode a previously reported β-spectrin from clustered acetylcholine receptors. Similarly for elf, the missing domains may be replaced through alternate exon usage to generate proteins with unique functions. A function for elf thus appears to be in the assembly and maintenance of specific domains on the cell surface—towards establishing hepatocyte polarity and thus differentiation.

Spectrins have also been shown to be conserved throughout evolution and am developmentally regulated. These results demonstrate that in keeping with brain β-spectrin (β-G spectrin), elf is also expressed in a tissue and stage specific manner and is conserved throughout evolution (Hu et al., J. Biol. Chem. 267:18715-18722, 1992; Zimmer et al., Brain Res. 594-75-88, 1992; Leto et al, Mol. Cell Biol. 8:1-9, 1988). Elf expression occurs in a gradient-like manner and close examination of Brain β-G spectrin has demonsrated similar gradient patterns, suggesting that a sudden on-off phenomenon at specific time points is simplistic. That elf is maximally expressed at day 10-11 suggests that it has an important function at this time, which continues, although to a lesser extent, with the later stages. For instance, it is conceivable that elf by conferring cell polarity mark the first overt sign of hepatocyte differentiation. Therefore, like Drosophila β-H spectrin, elf may play a role in facilitating a “velcro-like” joining of neighboring cell membranes as they extend (Thomas et al., Development 120:2039-2050, 1994). In this way elf may mark the polarization of the surrounding mesodermal cells, enabling foregut endodermal cells to invade this area and differentiate into hepatocytes. Molecular markers have been invaluable in the dissection of inductive events in embryological studies (New et al., Curr. Opin. Genet. Dev. 1:196-203, 1991; Sive, Genes Dev. 7:1-12, 1993). For instance, in Xenopus, Epi 1, an antibody specific for epidermis, has been used to elucidate the role of the blastopore lip in the neural induction process (Savage et al., Dev. Biol. 133:157-168, 1989). Similarly activins (regulating keratin) (Asashima et al., P.N.A.S. 88:6511-6514, 1991), vg-1 (Thomsen et al., Cell 63:485-493, 1990) and other genes belonging to the TGF-β family, as well as wnt and bFGF families represent components of the cascade leading to the commitment to particular mesodermal fate. For instance, vg-1, originally isolated by differential screening is to cells inducing embryonic mesoderm, the posttranslational processing of Vg-I precursor protein on the future side of the embryo being a key step in generating dorsal mesoderm and body axis in Xenopus (Thomsen et al., Cell 74:433-441, 1993). Similarly, in isolating putative inductive agents required for liver formation, a key step is the identification of mRNAs localized to cardiac/liver mesenchyme: elf and its regulatory genes will help to elucidate this area.

More recently, cell-cell interactions have been shown to be important for several cell fate decisions. In C. elegans for instance, lin-12 and glp-1 have been shown to encode transmembrane proteins mediating intracellular communication, and are required for the specification of several anterior fates. In Drosophila, the establishment of secondary epithelia which are the result of a mesenchymal-epithelial transition, is thought to be dependent upon two separate adhesions systems: direct interactions between the developing midgut endoderm and the visceral mesoderm on one hand and, adhesive interactions between the epithelial cells themselves on the other. While the latter cell-cell interaction is thought to be controlled by shotgun, control of apicobasal polarity is thought to be caused by genes such as cnanbs and stardust (Tepass et al, Cell 61:787-799, 1990). Although it is known that the biogenesis of cell surface polarity in hepatocyte formation is an early event, implying that the mechanisms for sorting plasma membrane molecules are functional at an early point, genes involved in cell signaling leading to cell fate in liver development have not been defined to date. The identification of such genes would give tremendous insight into the cell-cell interactions involved in foregut endodermal cell migration and subsequent morphogenesis of the liver as an organ. These studies establish the principle that specific mesoderm mRNAs are localized in a way that guarantees their subsequent segregation to specific mesodermal tissue, in this case the presumed mesodermal component of the liver as demonstrated by embryonic explant studies (Le Douarin, 1975).

Cloning and Sequencing of Elf:

All embryonic liver was obtained from matings of random-bred ICR mice (Harlan). The plug date was designated as Day 0 and embryos collected at days 10.0, 11.5 and 12.5 p.c.; these were staged by morphological criteria (Theiler, The House Mouse, New York: Springer-Verlag, 1989). The livers were dissected, pooled and lysed. To prepare cDNA libraries, RNA was isolated (Chomczynski et al., Analyt. Biochem. 162:156-159, 1987) and poly(A)+ RNA selected using oligo(dT)-cellulose (Collaborative Research Type 3). 1 to 5 mg of poly(A)+RNA were used in the preparation of oligo(dT)—primed cDNA libraries. cDNA library construction of days 11.5 and 12.5 embryonic liver was carried out by conventional techniques (Gubler et al., Gene 25:263-269, 1983), and the day 10.0 and adult mouse liver using the Stratagene Unizap cDNA library kit. Two subtracted libraries were then constructed (Schweinfest et al., Genet. Anal. Tech. Appl. 7:64-70, 1990). The resulting subtracted libraries comprised 64 clones (11.5-12.5), and 110 clones (10.5-11.5). The process involved: (a) Biotinylation: fifty micrograms of cDNA from day 12.5 liver library at 10 mg/ml were biotinylated in HE buffer (10 mM Hepes, pH 7.5, 1 mM EDTA, Clontech Labs.); (b) Subtraction was then done by the streptavidin-phenol extraction: the streptavidin-biotin hybrid duplexes represent common gene products which selectively partition into the phenol interface, leaving the unique, subtracted single stranded cDNA in the aqueous phase. After synthesis of second strand DNA and overnight precipitation, one tenth of the DNA was used to transform competent XL Blue cells. Transformation using all the subtracted DNA led to the identification of 174 recombinant colonies. Purification of bacteriophages, preparation of DNA were carried out by the stratagene in vivo excision protocol. Plasmid DNA was sequenced using 77 DNA polymerase (Sanger et al., J. Mol. Biol. 143:161-178, 1980).

Sequence Analysis:

The NCBI non-redundant (nr) and EST databases were searched using the blastp2 and blastn2 programs, which permit gapped alignments (Altschul et al., Methods in Enzymology 256:460-480, 1996), with the default parameters and elf protein or nucleotide sequences as queries.

RNA Preparation and Analysis:

Embryos were collected at day 10.0, 11.5 and 12.5 p.c. Embryonic livers were dissected in Dulbecco's modified Eagle's medium (high glucose) and 20 mM Hepes pH 7.3. The livers for the specific stages were pooled and total RNA isolated (Chomczynski et al., supra). 10 micrograms of RNA were electrophoresed on a 1% formaldehyde gel and transferred onto Hi-bond nylon membrane (Amersham) using standard procedures (Sambrook et al., 1989, supra). Radioactive, ³²P-labeled probes were synthesized by random primer methods (Feinberg et al., Analyt. Biochem. 137:266-267, 1984) and hybridized to the Nylon filters. Filters were washed at high stringency with a final wash in 0.2×SSC (30 mM NaCl, 3 mM sodium citrate, pH 7.4) 0.5% Sodium Dodecyl Sulfate at 65° C. for 60 minutes. Filters for each probe were stripped and rehybridized with other probes to confirm that no cross hybridization signals were obtained under initial screening conditions. These filters were then autoradiographed with intensifying screens at −70° C.

In Situ Analysis:

In situ analysis was performed for elf (Cox et al., Dev. Bio. 100:197-206, 1989). The RNA probes were synthesized and labeled with ³⁵S-UTP (400 Ci/mmole) via the T7 or SP6 promoter for RNA polymerase. Sense or antisense probes were added to the appropriate sections, mounted, sealed with rubber cement and incubated at 50° C. overnight. After incubation, sections were washed with 50% formamide/5×SSC/10 mM DTT (50° C.; 2×30 min.) followed by 4×SSC/TE, incubated with RNase A (20 mg/ml) and RNase TI (500 U/ml; 37° C. 30 min), rinsed again with 4×SSC/TE (37° C., 30 min), twice 2x SSC (25° C., 15 min), twice in 0.1×SSC (25° C., 15 min), dehydrated with an ethanol series (containing 0.3 M ammonium acetate) and air dried. For autoradiography, slides were dipped in NTB 2 emulsion diluted 1:1 with 2% glycerol in water and dried. Exposure times were from @ weeks to four months. The emulsion was developed according to manufacturer's directions.

In Vitro Translation of Elf:

Bluescript containing elf was transcribed with T7 RNA polymerase using the in Vitro Eukaryotic Translation kit and MCAP mRNA Capping kit (Stratagene). The RNA transcript was translated in vitro into protein for 90 minutes in the presence of [³⁵S] methionine using nuclease-treated rabbit reticulocyte lysate (Promega) and run on 4% denaturing polyacrylamide gels.

Liver Explant Cultures:

Mouse embryos were obtained from Harlan ICR mice. The age of the embryos was determined by days post appearance of the vaginal plug (day 0). The embryos were further characterized by the number of somites. Isolation of mouse hepatic endoderm, liver buds and mesoderm (en bloc dissection) was as follows: during the 10th day of gestation, the liver bud becomes evident as a thickening of the ventral wall of the foregut, near the origin of the yolk stalk. This ventral endoderm was then either taken alone and cultured, or alternatively with the surrounding mesoderm: the portion of the embryo between the otocyst and the umbilical region. Organ culture: Embryos were placed into nucleopore filters in a humid chamber as described (Houssaint, 1980, supra) and cultured for 48 hours or 96 hours. Microscopy: The explants were fixed as in the in situ hybridization protocols, and RNA isolated as described above. 7 mm sections were stained with hematoxylin, eosin and periodic acid schiff (PAS) for glycogen, an indicator of differentiated hepatocytes. For RNA analysis, semiquantitative RT-PCR was performed as described in FIGS. 19 and 20. The invention contemplates the use of such liver explant culture for tissue engineered composites as a form of liver restoration therapy.

Immunohistochemical Characterization:

Antibody to a peptide corresponding to amino acids 145-157 (CLRRKYRSREQPQS) of praja1 (COVANCE), was used for immunohistochemical localization in liver explant cultures. Embryos were fixed and embedded into paraffin, sectioned and immunostained using indirect immunohistochemistry according to protocols routinely used (Schevach, Current protocols in immunology, Green Publishing Associates and Wiley Interscience, 1991). 8 μm sections were deparaffinized in xylene, the tissue rehydrated in graded alcohols, and rinsed in PBS. The sections were initially treated with a protease (0.1% Trypsin in 0.05 M PBS) and incubated at 37° C. for 30 min. Endogenous peroxide was then removed using 3% hydrogen peroxide. Sections were blocked in PBS containing 5% goat serum for 30 min. at room temperature. Sections were then incubated overnight at 4° C. in a Humidor with diluted rabbit anti-mouse antibody directed against the PRAJA1 peptide, all further steps were done at room temperature. Six 5 min. rinses with PBS-S were performed after each successive step. After incubation in the primary antisera, slides were washed six times for 5 min. in 1×PBS at room temperature. Sections were incubated with a second antibody (diluted in 0.05 M PBS in 1% serum) for 30 min. at room temperature.

After rinses the substrate was added as follows: Insoluble Peroxidase substrate DAB (Sigma Fast). 100-150 microliter substrate solution was added to cover the entire tissue on the slide. Color development was monitored under microscope. After rinsing in distilled water for 5 min, staining was performed with Harris hematoxylin solution modified (Sigma) for 1 min., followed by a rinse in distilled water for 5 min. Sections were dehydrated by passage through distilled water, then graded alcohol concentrations and finally xylene. Coverslips were mounted using DPX (Fluka labs) or Permount (Fischer scientific), before observation. For the negative controls only the primary antibody diluting solution was added, without any antibody.

Generation of Antibodies:

Peptide-specific rabbit anti-mouse polyclonal antibodies to sequences in the N-terminal and C-terminal of ELF3 were generated as described in Porter et al., J. Cell. Biol. 117:997-1005 (1992). The sequences of the synthetic peptides were ELQRT SSVSG PLS (residues 2 to 14 at N-terminus) for the preparation of EL-1, and FNSRR TASDH SWSGM (residues 2140 to 2154 at C-terminus) for the preparation of EL-2. IgG was isolated from antisera by Protein A/G column (Pharmacia) and applied to affinity columns to which the appropriate synthetic peptides had been covalently linked (Pharmacia). The columns were washed with several volumes of buffered saline and then eluted with Elution buffer (pH 2.8, Pharmacia). The eluted fractions were collected into tubes containing sufficient 1 M Tris-HCl, pH 8.0, to bring their pH to 7.2. Affinity-purified antibodies and the antibody fractions that failed to bind to the affinity column were dialyzed against buffered saline containing 10 mM NaN₃ and stored at 4° C.

The specificity of the antibodies was assesses by enzyme-linked immunosorbant assays (ELISA), following the method of Engvall (Methods Enzymol. 70:419-439, 1980), and by immunoblotting of the synthetic peptides separated by SDS-PAGE. The results from ELISA confirmed the specificity of the antibodies for their corresponding antigens, as did the immunoblotting.

EXAMPLE 3

In accordance with the present invention, genes such as the ones discussed above which are involved in growth and differentiation of hepatocytes will also be involved in liver repair. This is important because cirrhosis or end stage liver disease is: (1) the fourth most common cause of death in the U.S; (2) related to fibrosis and nodular hyperplasia; (3) an important risk factor for hepatocellular carcinoma; and (4) currently has no suitable medical treatment.

One such mode of treatment will be the use of the elf protein, such as the three isoforms elf 1-3 as set forth above. In FIG. 13, the ELF spectrin membrane skeleton is shown. Spectrins are rod shaped, alpha and beta subunits. Helix linked by short actin filaments at junctional complexes that include AE2, protein 4.1, myosin. This membrane skeleton attaches to the plasma membrane at 2 sites, by ankyrin and the beta subunit of spectrin.

In FIG. 14, a comparison of ELF3 and 1 to Beta general spectrin is shown. Spectrins have three domains: Domain I binds Actin; Domain II binds to ankyrin; and Domain III dimerizes spectrin and gives tissue specificity. Sharp differences occur at the amino and C terminal ends. ELF1 differs from ELF3 in that it does not have an ankyrin binding domain.

Further studies have shown that the functional role of ELF may be associated with the ankyrin binding domain. Through antisense oligonucleotides to the ankyrin binding domain, ANK1 inhibits membrane associated ELF3. In addition, the inhibition of the ANK binding domain of ELF3 may result in the loss of intrahepatic bile ducts. This has a phenotype similar to Primary Biliary Cirrhosis (PBC): a disease of unknown etiology resulting in the destruction of Small and Medium sized intrahepatic Bile Ducts.

ELF has a distinct pattern of ELF expression in primary biliary cirrhosis, such as shown in FIG. 15. In panel A—a decrease in membrane labeling of ELF in early PBC is shown. In panels B and D—moderately advanced PBC is shown along with an absence of membrane labeling with a concomitant increase in cytoplasmic labeling of ELF. Panel C shows the absence in ELF labeling in fibrotic cirrhotic PBC tissue, lacking hepatocytes.

Accordingly, the evidence shows that ELF1 and 3 are Beta Spectrins expressed in bile duct epithelial cells and hepatocytes. In addition, ELF3 (membrane spectrin) inhibition leads to loss of intrahepatic bile ducts in explant cultures, with increased presence of lymphocytes. Decreased membrane labeling of ELF and marked increase in cytoplasmic labeling characterize PBC.

Further, antisense studies appear to show a primary role for ELF in PBC. Antibody studies in PBC support the role of ELF in its pathogenesis. Abnormal distribution of ELF may affect intracellular trafficking and may explain changes in AE2 expression and the resulting cholestasis seen in PBC.

Since the above reflects the mechanism by which ELF disruption results in primary biliary cirrhosis, investigations were made as to whether there were other pivotal protein interactions involved. Recent studies in smad2/3 mutants suggested the involvement of the TGF Beta pathway in PBC and the ultimate use of the elf proteins of the present invention in treating or preventing PBC and other liver diseases.

Transforming growth factor-β(TGF-β)is the major cytokine involved in organ fibrosis. It inhibits growth of hepatocytes and some hepatocellular carcinomas (HCC). The SMAD proteins serve as intracellular mediators of TGF-β and activins. TGF-Beta receptor activation involves phosphorylation of SMAD2 and SMAD3 and heteromeric complexes with SMAD4, such as shown in FIG. 16. Complexes translocate to the nucleus to control expression of target genes.

Studies that shown that animals lacking smad2 die before 8.5 days of development (E8.5), and thus smad2 is required for gastrulation and mesoderm induction. Animals lacking smad3 are viable but suffer mucosal immunodeficiency.

To investigate potential genetic cooperactivity in the smad gene family, we intercrossed strains lacking smad2 and smad3. These studies showed that (1) Mice doubly heterozygous for disruptions of the smad2 and smad3 genes display novel phenotypes not present in either single heterozygous; (2) Mutants die at E14 with marked liver hypoplasia; (3) 1-2% of smad2+/− smad3+/− animals survived to weaning; and (4) Mutant livers occur in 30-40% of the wild type.

Immunohistochemical analysis was conducted of hypoplastic liver sections using antibody to alpha-fetoprotein, and the results are shown in FIG. 17. In FIG. 17, Panel A shows wild type liver sections, cords of well organized and differentiated embryonic hepatocytes. Early primitive bile ducts are seen. On the other hand, Panel B of FIG. 17 shows smad2/3 mutant embryos, where hepatocytes are present, but normal cell architecture is lost. In addition, there is an absence of cell plates, and only primitive bile ducts.

Explant embryonic liver from mutants were cultured with HGF in an attempt to rescue the mutant embryos. When mutant embryonic livers were cultured in the presence of cardiac mesoderm, severe apoptosis occurred as seen in FIG. 18b, compared to wild type cultures in FIGS. 18a and 19 a with good hepatic growth.

Mutants cultured in the presence of HGF (5/50 ng/mL) show a rescue of the hepatic phenotype, with the formation of well differentiated hepatocytes, as well as primitive bile ducts as seen in FIG. 19c.

These tests also showed that smad2/smad3 mutant embryos die at day 14 with profound anemia and liver hypoplasia, and hepatic stem cell proliferation in the smad mutants is dramatically reduced. Hepatocyte and erythrocyte differentiation in the smad mutants is reduced by 10%, and HGF can rescue the hepatic phenotype in explant cultures.

Accordingly, it appears that Smad 2 and 3 are essential for hematopoisis and growth of developing liver. Gut and hepatic lineage are not altered in smad mutant mice. Smad 2 and 3 thus appear to be required for hepatic stem cell proliferation and cytoskeletal organization.

Moreover, ELF inhibition results in a phenotype with features suggestive of primary biliary cirrhosis (PBC). ELF expression is identical in smad2/3 mutants and PBC. It thus appears that smad2/3 is involved in the pathogenesis of PBC. Evidence for this includes the supression of smad3 in PBC; Smad2 nuclear localization absent in PBC; Smad2 and 3 bind to ELF in PBC tissue; Smad2/3 mutants have a severe but similar phenotype seen in primary biliary cirrhosis; and cytoskeletal protein interactions with ELF spectrins play a pivotal role in the pathogenesis of primary biliary cirrhosis.

It thus appears that genes such as the elf 1,3 proteins of the present invention and smads 2,3 which are involved in growth and differentiation of hepatocytes will be involved in liver repair seen in diseases such as primary biliary cirrhosis.

In summary, transforming growth factor-β (TGF-β) is a major cytokihe involved in multiple cellular processes including differentiation, proliferation, migration, fibrosis and apoptosis, and SMAD proteins serve as intracellular signaling molecules of TGF-β and activins. In novel phenotypes with smad2/3 intercrosses, almost all mutants died at E14 with marked liver hypoplasia and loss of primitive bile ducts. The smad2/3 mutants were notable for a marked fall in the expression of elf3, β-spectrin in accordance with the present invention. Antisense studies to elf3 and studies in tissue from patients with primary biliary cirrhosis suggest that a crucial role for ELF in this disorder and intrahepatic bile duct formation. In addition, smad2/3 mutants have a severe but similar phenotype seen in primary biliary cirrhosis. SMAD2, and SMAD3 bind to ELF3. These results taken together suggest that elf gene is an important player in intrahepatic bile duct formation and cirrhosis, and this process involves Smad2 and Smad3.

In short, ELF interactions with TGF Beta signaling molecules Smad2 and 3 are crucial for bile duct formation, as well as cirrhotic conditions such as PBC.

EXAMPLE 4

In our search for genes that are involved in liver repair, we utilized a specific cloning strategy of subtractive hybridization with embryonic liver cDNA libraries, and identified two novel Beta Spectrins termed elf(4), such as disclosed above. Antisense studies utilizing cultured liver explants show a vital role of elf3 in hepatocyte differentiation and intrahepatic bile duct formation, and to be a marker in cirrhosis. A similar loss of intrahepatic bile ducts is noted in smad2/3 knockouts. Notably ELF expression is diminished in these knockouts. Furthermore, we have shown that ELF binds directly and specifically to TGF Beta signaling molecules Smad2 and Smad3. Together, these results suggest a model in which ELF interactions with Smad2 and Smad3 are pivotal for bile duct formation.

These results suggest that ELF interactions with TGF Beta signaling molecules Smad2 and 3 are crucial for bile duct formation, as well as cirrhotic conditions such as PBC. Experiments performed on these molecules have given initial information of the mechanisms associated with cirrhosis and repair and how ELF and the TGF Beta signaling pathway activate these regulatory mechanisms.

These results demonstrate that elf is expressed as four transcripts in the liver, a 9.0 Kb primary transcript and three secondary transcripts (5 Kb, 4.0 Kb and 2.4 Kb). In addition, studies have shown an interaction between endogenous ELF and Smad2. For this, ELF was immunoprecipitated from liver lysates and HepG2 cells using an affinity-purified anti-ELF polyclonal antibody, and Smad2 and 3 were visualized by immunoblotting with anti-Smad2 and anti-Smad3 antibody. In immunoprecipitates prepared with preimmune antisera, no Smad2 was detectable. However, in the anti-ELF immunoprecipitates, we could clearly detect Smad2 and 3 coprecipitating with ELF. Together, these results demonstrate that ELF is a specific partner for receptor-regulared Smads 2 and 3 in the TGFβ/activin signaling pathway. Our biochemical analyses of ELF/Smad2 and Smad3 interactions suggest that ELF functions upstream in the pathway and might control the subcellular localization of Smad2 and Smad3.

Other important information of relevance to the usefulness of the elf proteins of the present invention relates to the following compounds or factors:

Transforming Growth Factor-Beta (TGF-Beta)

Transforming growth factor-Beta (TGF-Beta) represents an extensive family of growth and differentiation factors including activin/inhibins and bone morphogenetic proteins (BMPs) (Heldin et al., 1997), that mobilize a complex signaling network to control cell fate by regulating proliferation, differentiation, motility, adhesion, and apoptosis. TGF-Beta promotes the assembly of a cell surface receptor complex composed of type I (TbRI) and type II (TbRII) receptor serine/threonine kineses. In response to TGF-Beta binding, TbRII recruits and activates TbRI through phosphorylation of the regulatory GS-domain. Activated TbRI then initiates cytoplasmic signaling pathways to produce cellular responses. SMAD proteins together comprise a unique signaling pathway with key roles in signal transduction by TGF-Beta and related factors. The founding member of the SMAD family, Mothers against dpp (Mad) was identified as a dominant enhancer of weakly mutant alleles of decapentaplegic, a BMP homologue in Drosophila melanogaster (Raftery et al., 1995; Sekelsky et al., 1995). Genetic screens in Caenorhabditis elegans for mutant phenotypes like those observed for Ser/Thr kinase receptors daf-1 and daf-4 revealed three genes, sma-2, sma-3 and sma-4, with homology to Mad (Savage et al., 1996). At present, nine vertebrate SMADs have been identified (Attisano and Wrana, 1998). They are characterized by homologous regions at their N- and C-termini known as Mad homology (MH)-1 and MH-2 domains, respectively.

Three classes of Smads with distinct functions have been defined: the receptor-regulated Smads, which include Smad1, 2, 3, 5, and 8; the common mediator Smad, Smad4; and the antagonistic Smads, which include Smad6 and 7 (Heldin et al., 1997; Attisano and Wrana, 1998; Kretzschmar and Massague, 1998). Receptor-regulated Smads (R-Smads) act as direct substrates of specific type I receptors, and the proteins are phosphorylated on the last two serines at the carboxyl terminus within a highly conserved SSXS motif (Macias-Silva et al., 1996; Abdollah et al., 1997; Kretzschmar et al., 1997; Liu et al., 1997b; Souchelnytskyi et al., 1997). Regulation of R-Smads by the receptor kinase provides an important level of specificity in this system. Thus, Smad2 and Smad3 are substrates of TGFβ or activin receptors and mediate signaling by these ligands (Macias-Silva et al., 1996; Liu et al., 1997b; Nakao et al., 1997), whereas Smad1, 5 and 8 are targets of BMP receptors and propagate BMP signals (Hoodless et al., 1996; Chen et al., 1997b; Kretzschmar et al., 1997; Nishimura et al., 1998). Once phosphorylated, R-Smads associate with the common Smad, Smad4 (Lagna et al., 1996; Zhang et al., 1997), and mediate nuclear translocation of the heteromeric complex. In the nucleus, Smad complexes then activate specific genes through cooperative interactions with DNA and other DNA-binding proteins such as FAST1, FAST2, and Fos/Jun (Chen et al., 1996, 1997a; Labbe et al., 1998, Zhang et al., 1998; Zhou et al., 1998). In contrast to R-Smads and Smad4, the antagonistic Smads, Smad6 and 7, appear to function by blocking ligand-dependent signaling (reviewed in Heldin et al., 1997).

Genetic analysis in Drosophila melanogaster and Caenorhabditis elegans, as well as TbRII and SMAD mutations in human tumors, emphasizes their importance in TGF-Beta signaling. Collectively, these factors constitute a communication network exploited by TGF-Beta family members to regulate gene expression, and suggest a paradigm in which signaling pathways activated by ligand binding and operating in parallel, converge at target promoters to produce ligand specific responses.

Receptor Interacting Proteins

Proximal signaling events coupling TGF-Beta receptor activation to biological responses involves proteins, such as FKBP12, Drosophila inhibitor of apoptosis (DIAP)-1 and -2 (Oeda et al., 1998), and TbRI associated protein (TRAP)-1 and -2 (Charng et al., 1998). The WD-domain protein TRIP-1 and TbRII (Chen et al., 1995), that directly bind the receptor complex, FKBP12, a binding protein for the macrolide immunosuppressant FK506, interacts with a Leu-Pro motif in the GS-domain of TbRI and other type I receptors (Wang et al., 1996, Chen et al., 1997). (Wang et al. 1996). Phosphorylation of R-Smads by the type I receptor is essential for activating the TGFβ signaling pathway (Heldin et al., 1997; Attisano and Wranga, 1998; Kretzschmar and Massague, 1998). However, little is known of how Smad interaction with receptors is controlled. Recently, a novel Smad2/Smad3 interacting protein that contains a double zinc finger, or PYVE domain, and has been identified called SARA; SARA recruits Smad2 into distinct subcellular domans and that SARA colocalizes and interacts with TGFβ receptors. TGFβ signaling induces dissociation of Smad 2 from SARA with concomitant formation of Smad2/Smad4 complexes and nuclear translocation. Moreover, deletion of the FYVE domain in SARA causes mislocalization of Smad2 and inhibits TGFβ-dependent transcriptional responses. Thus, SARA defines a component of TGFβ signaling that functions to recruit Smad2 to the receptor by controlling the subcellular localization of Smad.

SMAD Domain Functions and Regulation by Intrinsic and Extrinsic Mechanisms

The MH2 region of pathway-restricted SMADs is involved in protein-protein interactions, particularly with other transactivating factors, such as interaction between Smad2 and the winged-helix transcription factor FAST-1 (Chen et al., 1996). Similarly, between Smad3 and the transcriptional coactivator CBP/p300 (Fen et al., 1998; Janknect et al., 1998). Additionally, the MH2 domains are responsible for homomeric and heteromeric interactions between SMADs (Zhang et al., 1997).

SMAD proteins reside in the cytoplasm, and upon stimulation translocate to the nucleus as part of an oligomeric complex (Attisano and Wrana, 1998). The observation that MH1 domain deletion from Smad2 results in constitutive nuclear localization (Baker and Harland, 1996) suggests an intrinsic inhibitory role for the MHI domain in signaling by pathway-restricted SMADs. Intrinsic inhibition of SMAD function mediated by the MH1 domain is relieved by agonist induced phosphorylation of the -SSXS motif, which presumably antagonizes the intramolecular MH1-MH2 interaction (Eppert et al., 1996; Schutte et al., 1996).

The MH1 domain is also involved in direct DNA binding. The MH1 domain Mad is necessary and sufficient for binding to the “quadrant” enhancer of the vestigal (vg) wing patterning gene in D. melanogaster (Kim et al., 1997). Similarly, Mad binds to the Dpp response element in the Ultrabithorax (ubx) promoter via its MH1 domain. Direct interaction of Smad3 and Smad4 with a CAGA-box, a DNA element repeated three times in the TGF-Beta responsive regions of the plasminogen activator inhibitor (PAI)-1 promoter has also been shown. This interaction requires the MH1 domain of both Smads. Smad3 additional requires either agonist stimulation or MH2 domain deletion. In a basal state, MH1 and MH2 domains provide intrinsic, reciprocal inhibition that is liberated by receptor activation and -SSXS phosphorylation.

Involvent of extrinic regulatory pathways such as the ERK MAP kinase pathway also contribute to SMAD regulation. In resonse to mitogenic growth factors such as epidermal growth factor (EGF) that signal through receptor tyrosine kinases (Denhardt, 1996), ERK-mediated phosphorylation of target transcriptional regulators contributes to the mitogenic influence of these factors. Recently, multiple serine residues in the linker region of Smad1 were shown to be phosphorylated by ERK, both in vitro and in vivo in response to EGF (Kretzschmar et al., 1997b). While phosphorylation of Smad1 by ERK was independent of -SSXS phosphorylation and did not effect association with Smad4, it did antagonize nuclear translocation of the SMAD oligomeric complex in response to BMP stimulation. ERK mitogenesis may involve simultaneous potentiation of growth promoting pathways and attenuation of growth inhibitory pathways.

In addition, Ca2+ dependent interaction between calmodulin (CaM) and several SMAD family members has been described (Zimmerman et al., 1998). CaM bound the N-terminal half of Smad2 between residues 76 and 208. Both CaM coexpression and a CaM-specific antagonist suggested a negative regulatory role for CaM in both activin and TGF-Beta signaling in a transient assay. Given the wide array of factors regulated by CaM either directly via protein-protein interaction or indirectly by CaM-dependent kineses, it is attractive to speculate that CaM influences SMAD protein function in response to agents that regulate intracellular Ca2+ flux.

Finally, other groups of proteins in which mammalian homoloques may be important in liver formation due to interactions with the ELF proteins of the present invention are the family of growth factors produced by the fat body and which are active on Drosophila imaginal disc cells, such as disclosed in Kawamura et al., Development 126:211-219 (1999), incoporated herein by reference.

28 1 5434 DNA Mus musculus misc_feature n=(a or c or t or g) 1 tcgggaaang attgatttgg ccncctcggn aaggcntttt attttgcnnc aaggagggcc 60 cggggggttt ccaaccnaaa taaaattttt tttcggatcc cgggggtttc ctcagggagt 120 tggggaattt tactttgaaa gcagatnttt cngagntccg ggtagctntc caataactnt 180 ttgtcatcat tgccagacgg cagatcaagg atgccttcgg tttacccgtg ctgttcagag 240 aacggctttt ggaagattga ttttaagtta tttaacagtc acagacaggt gtcatntntg 300 gagaatagag gcaagtccgc ggtgagggat gaagcaggag agattagggg aaggcagaca 360 ggactgctgg gccaaggaag ctgtgctgat ttgagcacag tgggaattca cgtacgcaat 420 ttcaaaggct ttagtggtaa attctgaagc tcagatgcag gcaagaccca agaggatagt 480 gtacacagag agaagagggt cntcaggatc gtgcgtagag tggagagagc cccaaaggca 540 ggagggaaga gcctcagtga ttacttaggg atgagggaga gaagaaaaaa ggttcttgca 600 aggtgtgggg tcttccaaat tcaggagttc actgccatat agagaaggtg tagcgggtga 660 aaggggccat gtgatgagga tggcaagcaa ggctgtggcg cagatgacga gatgcctggg 720 tcgggaggtc aggggagacc caggattggg gtcacctgtg tctgcgcaga ggggaagcca 780 ccctgcaact ggcccagcac tgagtccaga ggaaaatgag gcagaggaca aaccagagct 840 tcggagacta agtgcaggta gggcgcgggc ggagcgtgag gagggcagcg gaccacgcga 900 gaggcctcga aggccaccgg acccgcgtcc gagagtctga gggccctgcc cacacctgcg 960 tggccccctc cccagaggcc acactccaag gccaccctag aacccgtctg tctgctcaag 1020 cccttgcaaa agacgtctgc gcagaggggg cgtggcaggc gtgctgtcac tcacggcctg 1080 ttagccaatc cacgagtgcg cccctccccg gagagggtgc gcggagggcc cgcccccgcc 1140 gccaccgcgg gtgtgaggag gccaggctgg cgcggctccc tccgcccggc agccttgcca 1200 ggtaaccggg ttcggcggga gggctggggg tcgcgcagcc ccctcgctcc ctgggaggcg 1260 tgcacactgc cgcggcgggt cccgtgtggg ccggaggccc gtgcgcgcgt cggaccgacg 1320 ggccgcagcc tgtgggcggg gttgcgtgcg tgacgggcgg ccgtgccccg cgttgtgtca 1380 ggcctgcgcg gggaaagctc ggccgaaccg aggtgtccag gtccgcccgc tgcggcctgc 1440 cccgggttgc ggggcgcagg cgcggcggtg ggcgggggtc gtccccagga gcgtctttgt 1500 tcccggcgcg ctgagggcgg agcctcaccc cgccccgccc ccgcgctcag tccccgcccc 1560 gcgtccgccc gcaggagctg ccaccgggtc ccgctggcct ccccggccgc cgccaccgcc 1620 tccgcctccg ccgctccggg cccgccggct tgcgtcgccg aggtcgctgc agc atg 1676 Met 1 gcg ggc gtc gcg acc ccc tgc gcc aac ggc tgc ggg cct ggc gca ccc 1724 Ala Gly Val Ala Thr Pro Cys Ala Asn Gly Cys Gly Pro Gly Ala Pro 5 10 15 tcc gaa gcc gag gtg ctg cac ctc tgc cgc agc ctc gag gtg ggc acc 1772 Ser Glu Ala Glu Val Leu His Leu Cys Arg Ser Leu Glu Val Gly Thr 20 25 30 gtc atg act ttg ttc tac tcc aag aag tcg cag cgg cca gaa cgg aag 1820 Val Met Thr Leu Phe Tyr Ser Lys Lys Ser Gln Arg Pro Glu Arg Lys 35 40 45 acc ttc cag gtc aag ttg gag acg cgc cag atc aca tgg agc cgc ggc 1868 Thr Phe Gln Val Lys Leu Glu Thr Arg Gln Ile Thr Trp Ser Arg Gly 50 55 60 65 gcg gac aaa atc gag ggg tcc agt aag tgc gcc cca ctc cgg cct gcc 1916 Ala Asp Lys Ile Glu Gly Ser Ser Lys Cys Ala Pro Leu Arg Pro Ala 70 75 80 tcg cgc ctg ccc gcc tcc caa aca ctt ggg caa act ttc ggg cct cgc 1964 Ser Arg Leu Pro Ala Ser Gln Thr Leu Gly Gln Thr Phe Gly Pro Arg 85 90 95 gcc tgg cgc ccc gtc tcc gcc cag tcc ctg gtg gtc act ctg ggg cgg 2012 Ala Trp Arg Pro Val Ser Ala Gln Ser Leu Val Val Thr Leu Gly Arg 100 105 110 gtg gag ggg ggc atc cgg gtc ttg gat cac ctg ata gga cac ccc ctc 2060 Val Glu Gly Gly Ile Arg Val Leu Asp His Leu Ile Gly His Pro Leu 115 120 125 ccc cag tag ggggggagtg ttccaggcac tttgccctga ggcctaagag 2109 Pro Gln 130 tcctcactgg ttggacaagt ggagtgggat tccggccctt agcatcgggc ggctgtcagt 2169 ggctgtgagg ggaagccaag acagggaccc cctcatccaa cctgagaacc tggggaaccg 2229 acaagatctt cctgcccact gccatttctc cagagtgtgc tgtctgtgaa aactcctaag 2289 agctccggga tgggcttatt ggcgcaagaa cctttggaat cctcatgtag aacttaggca 2349 gatgttgggg tagggctggt tgtgaagcag agccctactc atctcccctc ttctttggga 2409 ggatggggta tgaaagctaa aaccgtgact gcttccccct cccatgtccc gtggatgggt 2469 tttttttttt tttttttttg ccccagatct gaattttgga ggtccatggt gctaggcagc 2529 catccaaagc tagagccatg gctcctttgc ccttgcagca tataacaagg agcttgcatt 2589 cagaaaggtt ccctggcctt gggttttggg gtccagccct ttgtgttgga tgttctcgtg 2649 accacagggt agcccagagt tgctcctctg gtttcctgtc gtacccttcc caaacctgag 2709 tgtggtgggt ttacacacaa gtctctggtg ggagaagtaa gtcaggagtt ttgagaaacc 2769 tcggctcttt ctgatagtca ttttcctcgg tgtgaggcag gatgaggagt ctttgcaact 2829 ccaggctttg agatgtttct tacaagaacc cccaaagagt ctatggttga agggacctag 2889 cctaagagcc aggtctgtgt tagagaaggg ggggtggtgt caggaagtaa caacggagag 2949 aaggtcccac agatcttcct ggggatggtg tacatgtgtg tcgatgggtg aggagatgag 3009 gaggaaggaa ggtttctgtg gtaagacagc catcctcaac tacaaacttc aggtctgaca 3069 gaattggccc ttaaccatca ccagtgccca tcagccctgg cctccgctgg aagaacattt 3129 cagtgatttt cagtgttggg ggatggaact gcagacagtt ccggtagtcc tgagacatca 3189 ctcagacatc aggttgcagg catggcattt tacgtttgta gtatttcctg tgtttaagtg 3249 gtggcattag ttccccggta gctagctctt ggtaacagct gcactgtaaa ccgtgtgtgt 3309 agcccagtag tggaagatag ctatggtatt tgaagccagt gtgttagctg tacgtcaccc 3369 agccaggtgc tttccctctc ggagcctcgg ttcctctgta agttagcaga agtatattta 3429 ctataaatgg tcacttttgg aagtgagata gttggtgtaa agtaagcaaa ctaaatatgt 3489 aatagatgcg agcagagacg ttacagaagt ttaagaacca gttattagta gcagtagcta 3549 tggtagatgc ttgtcctcct agaccctggg atggggcttc tgagggaggt ctaatgtggc 3609 tgttagaaaa agaaagggct ctgagggagg agggccgaga gagggtcccg ttctccttaa 3669 ttgcattacc caggataaaa gaggaaactc ttgttttgcc gtacatcgtt tacccttctg 3729 ttcacctgtc atgtaagatg agtttctatg tttggaattt tgtacattgg atgccattgt 3789 gagttggggc ctggacagaa agaagggact tagagacaga accatccagt ccgttttgtc 3849 tcacttgggt ctttgaggat gggtggcagg aatacagagg acgtcacctt tccagaccca 3909 caaaagtcac ccagagatat gcatgttttc attgggcccg accctgtgat ttttggggtc 3969 cagaatgaag gctgcagact agcctgtgtg gacttcatac cttgtaaatg gagcccacca 4029 ccgaagccct gccccacttc tgctggaatg cacctcactg cctttgtggg ttcccaaacc 4089 tgcagcctcc tgcagattgt gaaaaggatt gagttgccag ctgggtccct actgtctggt 4149 ctcttgttca gatgcctcag gtatttgact ttttgctgat aaccttatcc ctacctgaag 4209 ccaggccaga gagaaagact gccgctgtct gccctcaggg tgctcacgga acacaacgac 4269 aggctgactg ccatttccta aatcttgagt tctctcactg tgacacctgt gaaactagtt 4329 agcaccttct gatgtctaag gcagcggtct acttgagaag tgctttggtg ctgtttggtt 4389 gtgtgactga agtcaggctg gtgtctggca tttatgttgc agaatttagt gagttaaaag 4449 cagccataga cttcctgccc agtgctaaac agacttttca ctctgctgca ggctagtcct 4509 cagaggactc tgctcccagg ttgtgttggt ggtaggcctt ggtctcctgt tttctgtagc 4569 ctttgttgcc ccttgtgaag agaaacctcc atgtttaggt ggtatttaca ggcagagacc 4629 tccatcttca tcaaagacgc cttcctaggc tttccatatg taatgcctgt agtgagatgg 4689 ctcagaccta ttcttcgtga ggttgtccag ttaaggacca ctgttggcat agtagctcca 4749 gtagagactc taaagctatg ttgttattgt ggtgaggatt gcagtaccaa ggggctggct 4809 ctgagagtag gtccgtggca cctaagaatt gtctgcacat gtccctcaag gattcctttt 4869 ngctggccca cagtgagaga gcagcagaaa gcatgcgcct ggatctaaga aaggttaatg 4929 aaaccatggt acctatggga gctttacaac ctgggcttct gtctccggta gccatttcta 4989 aaaganatta tgaaattgtg gtagattgaa agatgttcct tactattcct ttacatcctg 5049 aggatcacga aagatttgct ttcagtattc ctactattaa ttttaaagaa cctatgaaaa 5109 gatatcaatg gacagttctt ccacaaggca tggctaataa tcctacctta tgtcaaantt 5169 gtggcacaac cattcacctg tgagacacaa tgactatgac tactcntcnt gatgatgatg 5229 angatgatga gatgatgatg atgatgatga tgacacacan gatagagatg attctaangc 5289 ggaaanatcc cgactgcttt ncttaaaatt accnncctnc gaaaagatta aacccgaaag 5349 gtcaccgatc tatatttngt ttaantnata ccgtttccca aaattttncg gacctnaant 5409 ttnatcaatt ttgtntatgn tcccc 5434 2 131 PRT Mus musculus 2 Met Ala Gly Val Ala Thr Pro Cys Ala Asn Gly Cys Gly Pro Gly Ala 1 5 10 15 Pro Ser Glu Ala Glu Val Leu His Leu Cys Arg Ser Leu Glu Val Gly 20 25 30 Thr Val Met Thr Leu Phe Tyr Ser Lys Lys Ser Gln Arg Pro Glu Arg 35 40 45 Lys Thr Phe Gln Val Lys Leu Glu Thr Arg Gln Ile Thr Trp Ser Arg 50 55 60 Gly Ala Asp Lys Ile Glu Gly Ser Ser Lys Cys Ala Pro Leu Arg Pro 65 70 75 80 Ala Ser Arg Leu Pro Ala Ser Gln Thr Leu Gly Gln Thr Phe Gly Pro 85 90 95 Arg Ala Trp Arg Pro Val Ser Ala Gln Ser Leu Val Val Thr Leu Gly 100 105 110 Arg Val Glu Gly Gly Ile Arg Val Leu Asp His Leu Ile Gly His Pro 115 120 125 Leu Pro Gln 130 3 6960 DNA Mus musculus CDS (333)..(6794) 3 cctgcgtcct tcctcctttt cctccttccc tcctccctcc cgggtaattt atttctagct 60 tccaggcaag ggccacacaa ggaaggaaat ccacagggga ttagatgccg gggtggtaac 120 tccaccaggc taggttggac tctgcagcca acttcctatc agatcaccct gcacctattt 180 ccgacccgac cggaatgcga ctggcttgag gtccagccct ttcgcctggg cgggagcaga 240 gccgcggaag ctgcttggag ttggatgggg gtaggaaggg gctggagcgg gaatcctacg 300 atgcaactgg cctgggccta aggttgggca ta atg gag ttg cag agg aca tcc 353 Met Glu Leu Gln Arg Thr Ser 1 5 agc gtt tca ggg ccg ctg tcg ccg gcc tac acc ggg cag gtg cct tac 401 Ser Val Ser Gly Pro Leu Ser Pro Ala Tyr Thr Gly Gln Val Pro Tyr 10 15 20 aac tac aac caa ctg gag gga aga ttc aaa cag ctc caa gat gag cgt 449 Asn Tyr Asn Gln Leu Glu Gly Arg Phe Lys Gln Leu Gln Asp Glu Arg 25 30 35 gaa gct gta cag aag aag acc ttc acc aag tgg gtc aat tcc cac ctt 497 Glu Ala Val Gln Lys Lys Thr Phe Thr Lys Trp Val Asn Ser His Leu 40 45 50 55 gca aga gtg tcc tgc cga atc aca gac ctg tac acg gac ctt cga gat 545 Ala Arg Val Ser Cys Arg Ile Thr Asp Leu Tyr Thr Asp Leu Arg Asp 60 65 70 gga cgg atg ctc atc aag cta ctg gag gtc ctc tct gga gag agg ctg 593 Gly Arg Met Leu Ile Lys Leu Leu Glu Val Leu Ser Gly Glu Arg Leu 75 80 85 cct aaa ccc act aag gga cgg atg cgg atc cac tgt ctg gag aat gtc 641 Pro Lys Pro Thr Lys Gly Arg Met Arg Ile His Cys Leu Glu Asn Val 90 95 100 gac aag gct ctt caa ttc ctg aaa gag cag aga gtc cat ctt gag aac 689 Asp Lys Ala Leu Gln Phe Leu Lys Glu Gln Arg Val His Leu Glu Asn 105 110 115 atg ggc tcc cat gac att gtg gat gga aac cac cgg ctg acc ctc ggc 737 Met Gly Ser His Asp Ile Val Asp Gly Asn His Arg Leu Thr Leu Gly 120 125 130 135 ctc atc tgg aca att att ctg cgc ttc cag atc cag gat att agt gtg 785 Leu Ile Trp Thr Ile Ile Leu Arg Phe Gln Ile Gln Asp Ile Ser Val 140 145 150 gag act gaa gat aac aaa gag aaa aag tct gct aag gat gca ttg ctg 833 Glu Thr Glu Asp Asn Lys Glu Lys Lys Ser Ala Lys Asp Ala Leu Leu 155 160 165 ctg tgg tgc cag atg aag aca gct ggg tac ccc aat gtc aac att cac 881 Leu Trp Cys Gln Met Lys Thr Ala Gly Tyr Pro Asn Val Asn Ile His 170 175 180 aat ttc acc act agc tgg agg gat ggc atg gcc ttc aat gca ctg ata 929 Asn Phe Thr Thr Ser Trp Arg Asp Gly Met Ala Phe Asn Ala Leu Ile 185 190 195 cat aaa cat cgg cct gac ctg ata gat ttt gat aaa ctg aag aaa tct 977 His Lys His Arg Pro Asp Leu Ile Asp Phe Asp Lys Leu Lys Lys Ser 200 205 210 215 aat gca cac tac aat ctg cag aat gca ttt aac ctg gca gag cag cac 1025 Asn Ala His Tyr Asn Leu Gln Asn Ala Phe Asn Leu Ala Glu Gln His 220 225 230 ctt ggc ctc act aaa ctg tta gac cct gaa gat atc agt gtg gac cac 1073 Leu Gly Leu Thr Lys Leu Leu Asp Pro Glu Asp Ile Ser Val Asp His 235 240 245 cct gat gag aag tct atc atc aca tac gtg gtg act tac tac cac tac 1121 Pro Asp Glu Lys Ser Ile Ile Thr Tyr Val Val Thr Tyr Tyr His Tyr 250 255 260 ttc tcc aag atg aag gcc ttg gct gtc gaa gga aag cgc att gga aag 1169 Phe Ser Lys Met Lys Ala Leu Ala Val Glu Gly Lys Arg Ile Gly Lys 265 270 275 gtg ctt gat aat gct ata gaa aca gag aaa atg att gag aag tac gag 1217 Val Leu Asp Asn Ala Ile Glu Thr Glu Lys Met Ile Glu Lys Tyr Glu 280 285 290 295 aca ctt gct tct gac ctt ctg gag tgg att gaa caa acc atc atc atc 1265 Thr Leu Ala Ser Asp Leu Leu Glu Trp Ile Glu Gln Thr Ile Ile Ile 300 305 310 cta aac aac cgc aaa ttt gct aat tca ctg gtt ggg gtc caa cag cag 1313 Leu Asn Asn Arg Lys Phe Ala Asn Ser Leu Val Gly Val Gln Gln Gln 315 320 325 ctc caa gca ttc aac acg tac cgc aca gtg gag aaa cca cct aag ttt 1361 Leu Gln Ala Phe Asn Thr Tyr Arg Thr Val Glu Lys Pro Pro Lys Phe 330 335 340 act gag aag ggg aat ttg gag gtg ctc ctt ttc gcg att cag agc aag 1409 Thr Glu Lys Gly Asn Leu Glu Val Leu Leu Phe Ala Ile Gln Ser Lys 345 350 355 atg cga gcg aat aat cag aag gtc tac atg ccc cgc gag ggg aag ctc 1457 Met Arg Ala Asn Asn Gln Lys Val Tyr Met Pro Arg Glu Gly Lys Leu 360 365 370 375 atc tct gac atc aac aag gcc tgg gaa aga ctg gaa aaa gca gaa cat 1505 Ile Ser Asp Ile Asn Lys Ala Trp Glu Arg Leu Glu Lys Ala Glu His 380 385 390 gag aga gaa ctg gct ctg cgg aat gag ctc ata cgg cag gaa aaa ctg 1553 Glu Arg Glu Leu Ala Leu Arg Asn Glu Leu Ile Arg Gln Glu Lys Leu 395 400 405 gaa caa ctc gcc cga aga ttt gat cgc aag gca gct atg agg gag aca 1601 Glu Gln Leu Ala Arg Arg Phe Asp Arg Lys Ala Ala Met Arg Glu Thr 410 415 420 tgg ctg agt gaa aac cag cgt ctt gtg tct cag gac aac ttt gga ttt 1649 Trp Leu Ser Glu Asn Gln Arg Leu Val Ser Gln Asp Asn Phe Gly Phe 425 430 435 gac ctt ccc gct gtt gag gct gct acc aaa aaa cac gag gcc att gag 1697 Asp Leu Pro Ala Val Glu Ala Ala Thr Lys Lys His Glu Ala Ile Glu 440 445 450 455 aca gac atc gct gca tat gaa gaa cga gtt cag gcc gtg gtg gct gtg 1745 Thr Asp Ile Ala Ala Tyr Glu Glu Arg Val Gln Ala Val Val Ala Val 460 465 470 gcc agg gaa ctt gaa gcc gag aac tac cat gac atc aag cgc atc aca 1793 Ala Arg Glu Leu Glu Ala Glu Asn Tyr His Asp Ile Lys Arg Ile Thr 475 480 485 gcg agg aag gac aat gtc atc cgg ctc tgg gaa tac ttg ctg gaa ctg 1841 Ala Arg Lys Asp Asn Val Ile Arg Leu Trp Glu Tyr Leu Leu Glu Leu 490 495 500 ctc agg gcc agg agg cag cgt ctt gag atg aac ctg gga ttg caa aag 1889 Leu Arg Ala Arg Arg Gln Arg Leu Glu Met Asn Leu Gly Leu Gln Lys 505 510 515 ata ttc cag gaa atg ctt tat att atg gac tgg atg gat gaa atg aag 1937 Ile Phe Gln Glu Met Leu Tyr Ile Met Asp Trp Met Asp Glu Met Lys 520 525 530 535 gtg cta ttg ctg tct caa gac tat ggc aaa cac tta ctt ggt gtt gaa 1985 Val Leu Leu Leu Ser Gln Asp Tyr Gly Lys His Leu Leu Gly Val Glu 540 545 550 gac ctg tta cag aag cat gcc ctg gtt gaa gca gac att gca atc caa 2033 Asp Leu Leu Gln Lys His Ala Leu Val Glu Ala Asp Ile Ala Ile Gln 555 560 565 gca gag cgt gta aga ggt gtg aat gcc tct gcc cag aag ttt gca aca 2081 Ala Glu Arg Val Arg Gly Val Asn Ala Ser Ala Gln Lys Phe Ala Thr 570 575 580 gat ggg gaa ggc tac aag cca tgt gac ccc cag gta att cga gac cgt 2129 Asp Gly Glu Gly Tyr Lys Pro Cys Asp Pro Gln Val Ile Arg Asp Arg 585 590 595 gtt gcc cac atg gag ttc tgc tat caa gag ctt tgt cag ctg gct gcc 2177 Val Ala His Met Glu Phe Cys Tyr Gln Glu Leu Cys Gln Leu Ala Ala 600 605 610 615 gag cgt agg gct cgc ctg gaa gag tcc cgt cgc ctc tgg aag ttc ttc 2225 Glu Arg Arg Ala Arg Leu Glu Glu Ser Arg Arg Leu Trp Lys Phe Phe 620 625 630 tgg gag atg gca gaa gag gaa ggc tgg ata cga gag aag gaa aag atc 2273 Trp Glu Met Ala Glu Glu Glu Gly Trp Ile Arg Glu Lys Glu Lys Ile 635 640 645 ctg tcc tct gat gat tac ggg aaa gac ttg acc agt gtc atg cgc ctg 2321 Leu Ser Ser Asp Asp Tyr Gly Lys Asp Leu Thr Ser Val Met Arg Leu 650 655 660 ctg agc aag cac cgg gca ttt gag gat gag atg agt ggc cgt agt ggc 2369 Leu Ser Lys His Arg Ala Phe Glu Asp Glu Met Ser Gly Arg Ser Gly 665 670 675 cat ttt gag cag gcc att aaa gaa ggt gaa gac atg att gca gag gaa 2417 His Phe Glu Gln Ala Ile Lys Glu Gly Glu Asp Met Ile Ala Glu Glu 680 685 690 695 cac ttt gga tcg gaa aag atc cgt gag aga atc att tat atc cgg gag 2465 His Phe Gly Ser Glu Lys Ile Arg Glu Arg Ile Ile Tyr Ile Arg Glu 700 705 710 cag tgg gcc aac ctg gaa cag ctc tca gcc att agg aag aag cgc cta 2513 Gln Trp Ala Asn Leu Glu Gln Leu Ser Ala Ile Arg Lys Lys Arg Leu 715 720 725 gag gaa gcc tca tta ctg cac cag ttc cag gct gat gct gat gat att 2561 Glu Glu Ala Ser Leu Leu His Gln Phe Gln Ala Asp Ala Asp Asp Ile 730 735 740 gat gct tgg atg tta gat ata ctc aag att gtc tcc agc aat gat gtg 2609 Asp Ala Trp Met Leu Asp Ile Leu Lys Ile Val Ser Ser Asn Asp Val 745 750 755 ggc cat gat gag tac tcc acg cag tct ctg gtc aag aag cat aaa gat 2657 Gly His Asp Glu Tyr Ser Thr Gln Ser Leu Val Lys Lys His Lys Asp 760 765 770 775 gta gca gaa gag atc acc aac tgc agg ccc act att gac aca ctg cat 2705 Val Ala Glu Glu Ile Thr Asn Cys Arg Pro Thr Ile Asp Thr Leu His 780 785 790 gag caa gcc agt gcc ctt cca caa gca cat gca gag tct cca gat gtg 2753 Glu Gln Ala Ser Ala Leu Pro Gln Ala His Ala Glu Ser Pro Asp Val 795 800 805 aag ggc cgg ctg gca gga att gag gag cgc tgc aag gag atg gca gag 2801 Lys Gly Arg Leu Ala Gly Ile Glu Glu Arg Cys Lys Glu Met Ala Glu 810 815 820 tta aca cgg cta agg aag cag gct ctg cag gac acc ctg gcc ctg tac 2849 Leu Thr Arg Leu Arg Lys Gln Ala Leu Gln Asp Thr Leu Ala Leu Tyr 825 830 835 aag atg ttc agt gag gct gat gcc tgt gag ctc tgg att gac gag aag 2897 Lys Met Phe Ser Glu Ala Asp Ala Cys Glu Leu Trp Ile Asp Glu Lys 840 845 850 855 gag cag tgg ctc aac aac atg cag atc cca gag aag ctg gag gac ctg 2945 Glu Gln Trp Leu Asn Asn Met Gln Ile Pro Glu Lys Leu Glu Asp Leu 860 865 870 gaa gtc atc cag cac aga ttt gag agc cta gaa cca gaa atg aac aac 2993 Glu Val Ile Gln His Arg Phe Glu Ser Leu Glu Pro Glu Met Asn Asn 875 880 885 cag gct tcc cgg gtt gct gtg gtg aac cag att gca cgg cag ctg atg 3041 Gln Ala Ser Arg Val Ala Val Val Asn Gln Ile Ala Arg Gln Leu Met 890 895 900 cac aat ggc cac ccc agt gaa aag gaa atc aga gct cag caa gac aaa 3089 His Asn Gly His Pro Ser Glu Lys Glu Ile Arg Ala Gln Gln Asp Lys 905 910 915 ctc aac acg agg tgg agt cag ttc aga gaa ctg gtg gac agg aaa aag 3137 Leu Asn Thr Arg Trp Ser Gln Phe Arg Glu Leu Val Asp Arg Lys Lys 920 925 930 935 gat gct ctt ctg tct gcc ctg agc atc cag aac tac cac ctc gag tgc 3185 Asp Ala Leu Leu Ser Ala Leu Ser Ile Gln Asn Tyr His Leu Glu Cys 940 945 950 aat gaa acc aaa tcc tgc atc cgg gag aag acc aag gtc atc gag tct 3233 Asn Glu Thr Lys Ser Cys Ile Arg Glu Lys Thr Lys Val Ile Glu Ser 955 960 965 acc caa gac ctt ggc aat gac ctg gca ggt gtc atg gcc ctg cag tgc 3281 Thr Gln Asp Leu Gly Asn Asp Leu Ala Gly Val Met Ala Leu Gln Cys 970 975 980 aag ctg act ggc atg gaa cga gac ttg gta gcc att gag gcg aag ctg 3329 Lys Leu Thr Gly Met Glu Arg Asp Leu Val Ala Ile Glu Ala Lys Leu 985 990 995 agt gac ctg cag aaa gaa gct gag aag ctg gag tcc gag cac cct gac 3377 Ser Asp Leu Gln Lys Glu Ala Glu Lys Leu Glu Ser Glu His Pro Asp 1000 1005 1010 1015 cag gct caa gct atc ctg tct cgg ctg gcc gag atc agt gat gtg tgg 3425 Gln Ala Gln Ala Ile Leu Ser Arg Leu Ala Glu Ile Ser Asp Val Trp 1020 1025 1030 gag gaa atg aag aca acc ctg aag aac cga gag gcc tcc ctg gga gag 3473 Glu Glu Met Lys Thr Thr Leu Lys Asn Arg Glu Ala Ser Leu Gly Glu 1035 1040 1045 gcc agc aag ctg cag cag ttt ctg cgg gac ttg gac gac ttc cag tct 3521 Ala Ser Lys Leu Gln Gln Phe Leu Arg Asp Leu Asp Asp Phe Gln Ser 1050 1055 1060 tgg ctc tcc agg acc cag act gct atc gcc tca gag gac atg ccc aat 3569 Trp Leu Ser Arg Thr Gln Thr Ala Ile Ala Ser Glu Asp Met Pro Asn 1065 1070 1075 acc ctc act gag gca gag aag ctt ctc aca cag cac gag aat atc aaa 3617 Thr Leu Thr Glu Ala Glu Lys Leu Leu Thr Gln His Glu Asn Ile Lys 1080 1085 1090 1095 aat gag atc gac aat tat gag gaa gac tac cag aag atg cgg gac atg 3665 Asn Glu Ile Asp Asn Tyr Glu Glu Asp Tyr Gln Lys Met Arg Asp Met 1100 1105 1110 ggc gag atg gtc acc cag ggg cag act gat gcc cag tat atg ttt ctg 3713 Gly Glu Met Val Thr Gln Gly Gln Thr Asp Ala Gln Tyr Met Phe Leu 1115 1120 1125 cgg cag cgg ctg cag gcc tta gac act ggc tgg aat gag ctc cac aaa 3761 Arg Gln Arg Leu Gln Ala Leu Asp Thr Gly Trp Asn Glu Leu His Lys 1130 1135 1140 atg tgg gag aac agg caa aac ctc ctc tcc cag tcc cat gcc tac cag 3809 Met Trp Glu Asn Arg Gln Asn Leu Leu Ser Gln Ser His Ala Tyr Gln 1145 1150 1155 cag ttc ctt agg gac acc aaa caa gct gaa gct ttt ctt aat aac cag 3857 Gln Phe Leu Arg Asp Thr Lys Gln Ala Glu Ala Phe Leu Asn Asn Gln 1160 1165 1170 1175 gag tat gtt ttg gct cat act gaa atg ccc acc acc ctg gaa gga gct 3905 Glu Tyr Val Leu Ala His Thr Glu Met Pro Thr Thr Leu Glu Gly Ala 1180 1185 1190 gaa gca gcc att aaa aag cag gag gac ttc atg acc acc atg gat gcc 3953 Glu Ala Ala Ile Lys Lys Gln Glu Asp Phe Met Thr Thr Met Asp Ala 1195 1200 1205 aac gag gag aag atc aat gct gtt gtg gag act ggc cga aga ctg gtg 4001 Asn Glu Glu Lys Ile Asn Ala Val Val Glu Thr Gly Arg Arg Leu Val 1210 1215 1220 agc gat ggg aac atc aac tcc gac cgc atc cag gag aag gtg gac tct 4049 Ser Asp Gly Asn Ile Asn Ser Asp Arg Ile Gln Glu Lys Val Asp Ser 1225 1230 1235 att gac gac aga cac agg aag aat cga gaa gca gcc agt gaa ctt ctg 4097 Ile Asp Asp Arg His Arg Lys Asn Arg Glu Ala Ala Ser Glu Leu Leu 1240 1245 1250 1255 atg agg tta aag gac aac cgt gat cta cag aag ttc ctg caa gat tgt 4145 Met Arg Leu Lys Asp Asn Arg Asp Leu Gln Lys Phe Leu Gln Asp Cys 1260 1265 1270 caa gag ctg tcc ctc tgg atc aat gaa aag atg ctt aca gct caa gac 4193 Gln Glu Leu Ser Leu Trp Ile Asn Glu Lys Met Leu Thr Ala Gln Asp 1275 1280 1285 atg tct tat gat gaa gcc aga aat ctg cac agt aaa tgg tta aag cat 4241 Met Ser Tyr Asp Glu Ala Arg Asn Leu His Ser Lys Trp Leu Lys His 1290 1295 1300 caa gca ttt atg gcg gaa ctt gca tcc aac aaa gaa tgg ctt gac aaa 4289 Gln Ala Phe Met Ala Glu Leu Ala Ser Asn Lys Glu Trp Leu Asp Lys 1305 1310 1315 att gag aag gaa gga atg cag ctt att tca gaa aag cca gaa aca gaa 4337 Ile Glu Lys Glu Gly Met Gln Leu Ile Ser Glu Lys Pro Glu Thr Glu 1320 1325 1330 1335 gct gtg gta aag gaa aaa ctc act ggt tta cat aaa atg tgg gaa gtc 4385 Ala Val Val Lys Glu Lys Leu Thr Gly Leu His Lys Met Trp Glu Val 1340 1345 1350 ctt gaa tcc aca acc cag acc aag gcc cag cgg ctc ttt gat gca aat 4433 Leu Glu Ser Thr Thr Gln Thr Lys Ala Gln Arg Leu Phe Asp Ala Asn 1355 1360 1365 aag gct gag ctt ttc aca caa agc tgc gca gat ctt gac aaa tgg cta 4481 Lys Ala Glu Leu Phe Thr Gln Ser Cys Ala Asp Leu Asp Lys Trp Leu 1370 1375 1380 cat ggc ctg gag agc cag att caa tct gac gac tat ggc aaa gac ctt 4529 His Gly Leu Glu Ser Gln Ile Gln Ser Asp Asp Tyr Gly Lys Asp Leu 1385 1390 1395 acc agt gtc aat att ctt ctg aaa aag caa cag atg ctg gag aat cag 4577 Thr Ser Val Asn Ile Leu Leu Lys Lys Gln Gln Met Leu Glu Asn Gln 1400 1405 1410 1415 atg gaa gtt cgg aag aaa gag atc gag gaa ctg cag agc caa gcc cag 4625 Met Glu Val Arg Lys Lys Glu Ile Glu Glu Leu Gln Ser Gln Ala Gln 1420 1425 1430 gcg ctg agt cag gag ggg aag agc aca gat gag gtg gac agc aaa cgc 4673 Ala Leu Ser Gln Glu Gly Lys Ser Thr Asp Glu Val Asp Ser Lys Arg 1435 1440 1445 ctt act gtg cag acc aag ttc atg gag ctt ctg gag ccc ttg agt gag 4721 Leu Thr Val Gln Thr Lys Phe Met Glu Leu Leu Glu Pro Leu Ser Glu 1450 1455 1460 agg aag cat aac ctg tta gct tcc aag gag atc cat cag ttc aac agg 4769 Arg Lys His Asn Leu Leu Ala Ser Lys Glu Ile His Gln Phe Asn Arg 1465 1470 1475 gat gtg gag gac gaa atc cta tgg gtt ggc gag agg atg cct ttg gca 4817 Asp Val Glu Asp Glu Ile Leu Trp Val Gly Glu Arg Met Pro Leu Ala 1480 1485 1490 1495 act tcc aca gat cat ggc cat aac ctt caa act gtg cag ctg tta ata 4865 Thr Ser Thr Asp His Gly His Asn Leu Gln Thr Val Gln Leu Leu Ile 1500 1505 1510 aag aaa aac cag acc ctc cag aaa gaa atc cag gga cac cag cct cgt 4913 Lys Lys Asn Gln Thr Leu Gln Lys Glu Ile Gln Gly His Gln Pro Arg 1515 1520 1525 att gat gac atc ttt gag agg agt caa aac atc atc aca gat agc agc 4961 Ile Asp Asp Ile Phe Glu Arg Ser Gln Asn Ile Ile Thr Asp Ser Ser 1530 1535 1540 agc ctc aat gcc gag gct atc agg cag agg ctc gct gac ctg aag cag 5009 Ser Leu Asn Ala Glu Ala Ile Arg Gln Arg Leu Ala Asp Leu Lys Gln 1545 1550 1555 ctg tgg ggg ctc ctc att gag gaa act gag aaa cgc cat aga cgg ctg 5057 Leu Trp Gly Leu Leu Ile Glu Glu Thr Glu Lys Arg His Arg Arg Leu 1560 1565 1570 1575 gag gag gca cac aag gcg cag cag tac tac ttt gat gca gct gaa gcc 5105 Glu Glu Ala His Lys Ala Gln Gln Tyr Tyr Phe Asp Ala Ala Glu Ala 1580 1585 1590 gag gca tgg atg agt gaa cag gag ttg tac atg atg tct gag gaa aag 5153 Glu Ala Trp Met Ser Glu Gln Glu Leu Tyr Met Met Ser Glu Glu Lys 1595 1600 1605 gcc aag gat gag cag agt gct gtc tct atg ttg aaa aag cac cag att 5201 Ala Lys Asp Glu Gln Ser Ala Val Ser Met Leu Lys Lys His Gln Ile 1610 1615 1620 tta gag caa gct gtt gag gac tat gca gag aca gta cac cag ctc tcc 5249 Leu Glu Gln Ala Val Glu Asp Tyr Ala Glu Thr Val His Gln Leu Ser 1625 1630 1635 aag act agc cgg gcg ctg gtg gct gac agc cat ccc gaa agt gag cgt 5297 Lys Thr Ser Arg Ala Leu Val Ala Asp Ser His Pro Glu Ser Glu Arg 1640 1645 1650 1655 att agc atg cgg cag tca aag gtc gac aag ctg tat gct ggc ctg aag 5345 Ile Ser Met Arg Gln Ser Lys Val Asp Lys Leu Tyr Ala Gly Leu Lys 1660 1665 1670 gac ctt gct gag gag agg aga gga aaa ctt gat gag agg cac agg ctg 5393 Asp Leu Ala Glu Glu Arg Arg Gly Lys Leu Asp Glu Arg His Arg Leu 1675 1680 1685 ttc cag ctc aac aga gag gtg gat gac ctg gaa cag tgg atc gct gag 5441 Phe Gln Leu Asn Arg Glu Val Asp Asp Leu Glu Gln Trp Ile Ala Glu 1690 1695 1700 agg gaa gtg gtc gca ggc tcc cat gag ttg gga cag gac tat gag cat 5489 Arg Glu Val Val Ala Gly Ser His Glu Leu Gly Gln Asp Tyr Glu His 1705 1710 1715 gtc acg atg tta caa gaa cgg ttc cga gaa ttt gct cga gac aca gga 5537 Val Thr Met Leu Gln Glu Arg Phe Arg Glu Phe Ala Arg Asp Thr Gly 1720 1725 1730 1735 aac att ggg cag gag cgt gtg gat aca gtt aat aac atg gca gat gaa 5585 Asn Ile Gly Gln Glu Arg Val Asp Thr Val Asn Asn Met Ala Asp Glu 1740 1745 1750 ctc atc aac tct gga cat tca gat gct gcc acc att gct gag tgg aaa 5633 Leu Ile Asn Ser Gly His Ser Asp Ala Ala Thr Ile Ala Glu Trp Lys 1755 1760 1765 gat ggt ctc aat gaa gcc tgg gct gac ctc ctg gag ctc att gac aca 5681 Asp Gly Leu Asn Glu Ala Trp Ala Asp Leu Leu Glu Leu Ile Asp Thr 1770 1775 1780 aga aca cag att ctt gct gcc tca tat gaa ctt cat aag ttt tac cat 5729 Arg Thr Gln Ile Leu Ala Ala Ser Tyr Glu Leu His Lys Phe Tyr His 1785 1790 1795 gat gcc aag gag atc ttt ggc cga atc cag gac aaa cac aag aaa ctc 5777 Asp Ala Lys Glu Ile Phe Gly Arg Ile Gln Asp Lys His Lys Lys Leu 1800 1805 1810 1815 cct gag gag ctt gga aga gat caa aac act gtg gaa act tta cag aga 5825 Pro Glu Glu Leu Gly Arg Asp Gln Asn Thr Val Glu Thr Leu Gln Arg 1820 1825 1830 atg cac acc acc ttt gag cac gac atc caa gct ctg ggc act cag gtg 5873 Met His Thr Thr Phe Glu His Asp Ile Gln Ala Leu Gly Thr Gln Val 1835 1840 1845 agg cag ctg cag gag gat gca gct cgc ctc cag gca gcc tat gca ggg 5921 Arg Gln Leu Gln Glu Asp Ala Ala Arg Leu Gln Ala Ala Tyr Ala Gly 1850 1855 1860 gac aag gct gat gac atc cag aag cgt gag aat gag gtc ctg gaa gcc 5969 Asp Lys Ala Asp Asp Ile Gln Lys Arg Glu Asn Glu Val Leu Glu Ala 1865 1870 1875 tgg aag tcc ctg ctg gat gct tgt gag ggt cgc agg gtg cgg ctg gta 6017 Trp Lys Ser Leu Leu Asp Ala Cys Glu Gly Arg Arg Val Arg Leu Val 1880 1885 1890 1895 gac aca gga gac aag ttc cgc ttc ttc agc atg gtg cgt gac ctc atg 6065 Asp Thr Gly Asp Lys Phe Arg Phe Phe Ser Met Val Arg Asp Leu Met 1900 1905 1910 ctc tgg atg gaa gat gtc atc cgg cag atc gag gcc cag gag aaa cca 6113 Leu Trp Met Glu Asp Val Ile Arg Gln Ile Glu Ala Gln Glu Lys Pro 1915 1920 1925 cgg gat gtg tca tct gtt gaa ctg tta atg aat aat cat caa ggt atc 6161 Arg Asp Val Ser Ser Val Glu Leu Leu Met Asn Asn His Gln Gly Ile 1930 1935 1940 aaa gct gaa att gat gct cgt aat gac agc ttt aca gcc tgc att gag 6209 Lys Ala Glu Ile Asp Ala Arg Asn Asp Ser Phe Thr Ala Cys Ile Glu 1945 1950 1955 ctt ggg aaa tcc ctg ctg gca cgg aaa cac tat gct tct gag gag atc 6257 Leu Gly Lys Ser Leu Leu Ala Arg Lys His Tyr Ala Ser Glu Glu Ile 1960 1965 1970 1975 aag gaa aag tta ctg cag ctg aca gag aaa aga aaa gaa atg att gac 6305 Lys Glu Lys Leu Leu Gln Leu Thr Glu Lys Arg Lys Glu Met Ile Asp 1980 1985 1990 aag tgg gaa gac cgg tgg gag tgg tta aga ctg att ttg gag gtc cat 6353 Lys Trp Glu Asp Arg Trp Glu Trp Leu Arg Leu Ile Leu Glu Val His 1995 2000 2005 cag ttc tca agg gat gcc agt gtg gca gag gct tgg ctg ctt gga cag 6401 Gln Phe Ser Arg Asp Ala Ser Val Ala Glu Ala Trp Leu Leu Gly Gln 2010 2015 2020 gaa cca tac cta tcc agc cgt gaa att ggc cag agt gta gac gaa gtg 6449 Glu Pro Tyr Leu Ser Ser Arg Glu Ile Gly Gln Ser Val Asp Glu Val 2025 2030 2035 gag aag ctt att aag cgc cat gag gcg ttt gaa aag tct gca gcg acc 6497 Glu Lys Leu Ile Lys Arg His Glu Ala Phe Glu Lys Ser Ala Ala Thr 2040 2045 2050 2055 tgg gat gag aga ttc tct gct ctg gaa agg ctg aca acg ttg gag cta 6545 Trp Asp Glu Arg Phe Ser Ala Leu Glu Arg Leu Thr Thr Leu Glu Leu 2060 2065 2070 ctg gaa gtg cgc aga cag caa gag gaa gaa gaa aga aag agg cgg cca 6593 Leu Glu Val Arg Arg Gln Gln Glu Glu Glu Glu Arg Lys Arg Arg Pro 2075 2080 2085 cct tct ccg gac cca aac acg aag gtt tca gag gag gct gag tcc cag 6641 Pro Ser Pro Asp Pro Asn Thr Lys Val Ser Glu Glu Ala Glu Ser Gln 2090 2095 2100 caa tgg gat act tca aaa gga gac caa gtt tcc cag aat ggt ttg ccg 6689 Gln Trp Asp Thr Ser Lys Gly Asp Gln Val Ser Gln Asn Gly Leu Pro 2105 2110 2115 gct gag cag gga tct cca cgg gtt agt tac cgc tct caa acg tac caa 6737 Ala Glu Gln Gly Ser Pro Arg Val Ser Tyr Arg Ser Gln Thr Tyr Gln 2120 2125 2130 2135 aac tac aaa aac ttt aat agc aga cgg aca gcc agt gac cat tca tgg 6785 Asn Tyr Lys Asn Phe Asn Ser Arg Arg Thr Ala Ser Asp His Ser Trp 2140 2145 2150 tct gga atg tgaagttcac taccatttgt caagaaccac tctgtccaca 6834 Ser Gly Met tcctttgacc ttttggcttc cacgtcaccc agagtgttaa aatttttact taattcatag 6894 ctgtccttga tttcatattt gtttgcattt aatttatgtt tctttggatc ctcattgcct 6954 caaagc 6960 4 2154 PRT Mus musculus 4 Met Glu Leu Gln Arg Thr Ser Ser Val Ser Gly Pro Leu Ser Pro Ala 1 5 10 15 Tyr Thr Gly Gln Val Pro Tyr Asn Tyr Asn Gln Leu Glu Gly Arg Phe 20 25 30 Lys Gln Leu Gln Asp Glu Arg Glu Ala Val Gln Lys Lys Thr Phe Thr 35 40 45 Lys Trp Val Asn Ser His Leu Ala Arg Val Ser Cys Arg Ile Thr Asp 50 55 60 Leu Tyr Thr Asp Leu Arg Asp Gly Arg Met Leu Ile Lys Leu Leu Glu 65 70 75 80 Val Leu Ser Gly Glu Arg Leu Pro Lys Pro Thr Lys Gly Arg Met Arg 85 90 95 Ile His Cys Leu Glu Asn Val Asp Lys Ala Leu Gln Phe Leu Lys Glu 100 105 110 Gln Arg Val His Leu Glu Asn Met Gly Ser His Asp Ile Val Asp Gly 115 120 125 Asn His Arg Leu Thr Leu Gly Leu Ile Trp Thr Ile Ile Leu Arg Phe 130 135 140 Gln Ile Gln Asp Ile Ser Val Glu Thr Glu Asp Asn Lys Glu Lys Lys 145 150 155 160 Ser Ala Lys Asp Ala Leu Leu Leu Trp Cys Gln Met Lys Thr Ala Gly 165 170 175 Tyr Pro Asn Val Asn Ile His Asn Phe Thr Thr Ser Trp Arg Asp Gly 180 185 190 Met Ala Phe Asn Ala Leu Ile His Lys His Arg Pro Asp Leu Ile Asp 195 200 205 Phe Asp Lys Leu Lys Lys Ser Asn Ala His Tyr Asn Leu Gln Asn Ala 210 215 220 Phe Asn Leu Ala Glu Gln His Leu Gly Leu Thr Lys Leu Leu Asp Pro 225 230 235 240 Glu Asp Ile Ser Val Asp His Pro Asp Glu Lys Ser Ile Ile Thr Tyr 245 250 255 Val Val Thr Tyr Tyr His Tyr Phe Ser Lys Met Lys Ala Leu Ala Val 260 265 270 Glu Gly Lys Arg Ile Gly Lys Val Leu Asp Asn Ala Ile Glu Thr Glu 275 280 285 Lys Met Ile Glu Lys Tyr Glu Thr Leu Ala Ser Asp Leu Leu Glu Trp 290 295 300 Ile Glu Gln Thr Ile Ile Ile Leu Asn Asn Arg Lys Phe Ala Asn Ser 305 310 315 320 Leu Val Gly Val Gln Gln Gln Leu Gln Ala Phe Asn Thr Tyr Arg Thr 325 330 335 Val Glu Lys Pro Pro Lys Phe Thr Glu Lys Gly Asn Leu Glu Val Leu 340 345 350 Leu Phe Ala Ile Gln Ser Lys Met Arg Ala Asn Asn Gln Lys Val Tyr 355 360 365 Met Pro Arg Glu Gly Lys Leu Ile Ser Asp Ile Asn Lys Ala Trp Glu 370 375 380 Arg Leu Glu Lys Ala Glu His Glu Arg Glu Leu Ala Leu Arg Asn Glu 385 390 395 400 Leu Ile Arg Gln Glu Lys Leu Glu Gln Leu Ala Arg Arg Phe Asp Arg 405 410 415 Lys Ala Ala Met Arg Glu Thr Trp Leu Ser Glu Asn Gln Arg Leu Val 420 425 430 Ser Gln Asp Asn Phe Gly Phe Asp Leu Pro Ala Val Glu Ala Ala Thr 435 440 445 Lys Lys His Glu Ala Ile Glu Thr Asp Ile Ala Ala Tyr Glu Glu Arg 450 455 460 Val Gln Ala Val Val Ala Val Ala Arg Glu Leu Glu Ala Glu Asn Tyr 465 470 475 480 His Asp Ile Lys Arg Ile Thr Ala Arg Lys Asp Asn Val Ile Arg Leu 485 490 495 Trp Glu Tyr Leu Leu Glu Leu Leu Arg Ala Arg Arg Gln Arg Leu Glu 500 505 510 Met Asn Leu Gly Leu Gln Lys Ile Phe Gln Glu Met Leu Tyr Ile Met 515 520 525 Asp Trp Met Asp Glu Met Lys Val Leu Leu Leu Ser Gln Asp Tyr Gly 530 535 540 Lys His Leu Leu Gly Val Glu Asp Leu Leu Gln Lys His Ala Leu Val 545 550 555 560 Glu Ala Asp Ile Ala Ile Gln Ala Glu Arg Val Arg Gly Val Asn Ala 565 570 575 Ser Ala Gln Lys Phe Ala Thr Asp Gly Glu Gly Tyr Lys Pro Cys Asp 580 585 590 Pro Gln Val Ile Arg Asp Arg Val Ala His Met Glu Phe Cys Tyr Gln 595 600 605 Glu Leu Cys Gln Leu Ala Ala Glu Arg Arg Ala Arg Leu Glu Glu Ser 610 615 620 Arg Arg Leu Trp Lys Phe Phe Trp Glu Met Ala Glu Glu Glu Gly Trp 625 630 635 640 Ile Arg Glu Lys Glu Lys Ile Leu Ser Ser Asp Asp Tyr Gly Lys Asp 645 650 655 Leu Thr Ser Val Met Arg Leu Leu Ser Lys His Arg Ala Phe Glu Asp 660 665 670 Glu Met Ser Gly Arg Ser Gly His Phe Glu Gln Ala Ile Lys Glu Gly 675 680 685 Glu Asp Met Ile Ala Glu Glu His Phe Gly Ser Glu Lys Ile Arg Glu 690 695 700 Arg Ile Ile Tyr Ile Arg Glu Gln Trp Ala Asn Leu Glu Gln Leu Ser 705 710 715 720 Ala Ile Arg Lys Lys Arg Leu Glu Glu Ala Ser Leu Leu His Gln Phe 725 730 735 Gln Ala Asp Ala Asp Asp Ile Asp Ala Trp Met Leu Asp Ile Leu Lys 740 745 750 Ile Val Ser Ser Asn Asp Val Gly His Asp Glu Tyr Ser Thr Gln Ser 755 760 765 Leu Val Lys Lys His Lys Asp Val Ala Glu Glu Ile Thr Asn Cys Arg 770 775 780 Pro Thr Ile Asp Thr Leu His Glu Gln Ala Ser Ala Leu Pro Gln Ala 785 790 795 800 His Ala Glu Ser Pro Asp Val Lys Gly Arg Leu Ala Gly Ile Glu Glu 805 810 815 Arg Cys Lys Glu Met Ala Glu Leu Thr Arg Leu Arg Lys Gln Ala Leu 820 825 830 Gln Asp Thr Leu Ala Leu Tyr Lys Met Phe Ser Glu Ala Asp Ala Cys 835 840 845 Glu Leu Trp Ile Asp Glu Lys Glu Gln Trp Leu Asn Asn Met Gln Ile 850 855 860 Pro Glu Lys Leu Glu Asp Leu Glu Val Ile Gln His Arg Phe Glu Ser 865 870 875 880 Leu Glu Pro Glu Met Asn Asn Gln Ala Ser Arg Val Ala Val Val Asn 885 890 895 Gln Ile Ala Arg Gln Leu Met His Asn Gly His Pro Ser Glu Lys Glu 900 905 910 Ile Arg Ala Gln Gln Asp Lys Leu Asn Thr Arg Trp Ser Gln Phe Arg 915 920 925 Glu Leu Val Asp Arg Lys Lys Asp Ala Leu Leu Ser Ala Leu Ser Ile 930 935 940 Gln Asn Tyr His Leu Glu Cys Asn Glu Thr Lys Ser Cys Ile Arg Glu 945 950 955 960 Lys Thr Lys Val Ile Glu Ser Thr Gln Asp Leu Gly Asn Asp Leu Ala 965 970 975 Gly Val Met Ala Leu Gln Cys Lys Leu Thr Gly Met Glu Arg Asp Leu 980 985 990 Val Ala Ile Glu Ala Lys Leu Ser Asp Leu Gln Lys Glu Ala Glu Lys 995 1000 1005 Leu Glu Ser Glu His Pro Asp Gln Ala Gln Ala Ile Leu Ser Arg Leu 1010 1015 1020 Ala Glu Ile Ser Asp Val Trp Glu Glu Met Lys Thr Thr Leu Lys Asn 1025 1030 1035 1040 Arg Glu Ala Ser Leu Gly Glu Ala Ser Lys Leu Gln Gln Phe Leu Arg 1045 1050 1055 Asp Leu Asp Asp Phe Gln Ser Trp Leu Ser Arg Thr Gln Thr Ala Ile 1060 1065 1070 Ala Ser Glu Asp Met Pro Asn Thr Leu Thr Glu Ala Glu Lys Leu Leu 1075 1080 1085 Thr Gln His Glu Asn Ile Lys Asn Glu Ile Asp Asn Tyr Glu Glu Asp 1090 1095 1100 Tyr Gln Lys Met Arg Asp Met Gly Glu Met Val Thr Gln Gly Gln Thr 1105 1110 1115 1120 Asp Ala Gln Tyr Met Phe Leu Arg Gln Arg Leu Gln Ala Leu Asp Thr 1125 1130 1135 Gly Trp Asn Glu Leu His Lys Met Trp Glu Asn Arg Gln Asn Leu Leu 1140 1145 1150 Ser Gln Ser His Ala Tyr Gln Gln Phe Leu Arg Asp Thr Lys Gln Ala 1155 1160 1165 Glu Ala Phe Leu Asn Asn Gln Glu Tyr Val Leu Ala His Thr Glu Met 1170 1175 1180 Pro Thr Thr Leu Glu Gly Ala Glu Ala Ala Ile Lys Lys Gln Glu Asp 1185 1190 1195 1200 Phe Met Thr Thr Met Asp Ala Asn Glu Glu Lys Ile Asn Ala Val Val 1205 1210 1215 Glu Thr Gly Arg Arg Leu Val Ser Asp Gly Asn Ile Asn Ser Asp Arg 1220 1225 1230 Ile Gln Glu Lys Val Asp Ser Ile Asp Asp Arg His Arg Lys Asn Arg 1235 1240 1245 Glu Ala Ala Ser Glu Leu Leu Met Arg Leu Lys Asp Asn Arg Asp Leu 1250 1255 1260 Gln Lys Phe Leu Gln Asp Cys Gln Glu Leu Ser Leu Trp Ile Asn Glu 1265 1270 1275 1280 Lys Met Leu Thr Ala Gln Asp Met Ser Tyr Asp Glu Ala Arg Asn Leu 1285 1290 1295 His Ser Lys Trp Leu Lys His Gln Ala Phe Met Ala Glu Leu Ala Ser 1300 1305 1310 Asn Lys Glu Trp Leu Asp Lys Ile Glu Lys Glu Gly Met Gln Leu Ile 1315 1320 1325 Ser Glu Lys Pro Glu Thr Glu Ala Val Val Lys Glu Lys Leu Thr Gly 1330 1335 1340 Leu His Lys Met Trp Glu Val Leu Glu Ser Thr Thr Gln Thr Lys Ala 1345 1350 1355 1360 Gln Arg Leu Phe Asp Ala Asn Lys Ala Glu Leu Phe Thr Gln Ser Cys 1365 1370 1375 Ala Asp Leu Asp Lys Trp Leu His Gly Leu Glu Ser Gln Ile Gln Ser 1380 1385 1390 Asp Asp Tyr Gly Lys Asp Leu Thr Ser Val Asn Ile Leu Leu Lys Lys 1395 1400 1405 Gln Gln Met Leu Glu Asn Gln Met Glu Val Arg Lys Lys Glu Ile Glu 1410 1415 1420 Glu Leu Gln Ser Gln Ala Gln Ala Leu Ser Gln Glu Gly Lys Ser Thr 1425 1430 1435 1440 Asp Glu Val Asp Ser Lys Arg Leu Thr Val Gln Thr Lys Phe Met Glu 1445 1450 1455 Leu Leu Glu Pro Leu Ser Glu Arg Lys His Asn Leu Leu Ala Ser Lys 1460 1465 1470 Glu Ile His Gln Phe Asn Arg Asp Val Glu Asp Glu Ile Leu Trp Val 1475 1480 1485 Gly Glu Arg Met Pro Leu Ala Thr Ser Thr Asp His Gly His Asn Leu 1490 1495 1500 Gln Thr Val Gln Leu Leu Ile Lys Lys Asn Gln Thr Leu Gln Lys Glu 1505 1510 1515 1520 Ile Gln Gly His Gln Pro Arg Ile Asp Asp Ile Phe Glu Arg Ser Gln 1525 1530 1535 Asn Ile Ile Thr Asp Ser Ser Ser Leu Asn Ala Glu Ala Ile Arg Gln 1540 1545 1550 Arg Leu Ala Asp Leu Lys Gln Leu Trp Gly Leu Leu Ile Glu Glu Thr 1555 1560 1565 Glu Lys Arg His Arg Arg Leu Glu Glu Ala His Lys Ala Gln Gln Tyr 1570 1575 1580 Tyr Phe Asp Ala Ala Glu Ala Glu Ala Trp Met Ser Glu Gln Glu Leu 1585 1590 1595 1600 Tyr Met Met Ser Glu Glu Lys Ala Lys Asp Glu Gln Ser Ala Val Ser 1605 1610 1615 Met Leu Lys Lys His Gln Ile Leu Glu Gln Ala Val Glu Asp Tyr Ala 1620 1625 1630 Glu Thr Val His Gln Leu Ser Lys Thr Ser Arg Ala Leu Val Ala Asp 1635 1640 1645 Ser His Pro Glu Ser Glu Arg Ile Ser Met Arg Gln Ser Lys Val Asp 1650 1655 1660 Lys Leu Tyr Ala Gly Leu Lys Asp Leu Ala Glu Glu Arg Arg Gly Lys 1665 1670 1675 1680 Leu Asp Glu Arg His Arg Leu Phe Gln Leu Asn Arg Glu Val Asp Asp 1685 1690 1695 Leu Glu Gln Trp Ile Ala Glu Arg Glu Val Val Ala Gly Ser His Glu 1700 1705 1710 Leu Gly Gln Asp Tyr Glu His Val Thr Met Leu Gln Glu Arg Phe Arg 1715 1720 1725 Glu Phe Ala Arg Asp Thr Gly Asn Ile Gly Gln Glu Arg Val Asp Thr 1730 1735 1740 Val Asn Asn Met Ala Asp Glu Leu Ile Asn Ser Gly His Ser Asp Ala 1745 1750 1755 1760 Ala Thr Ile Ala Glu Trp Lys Asp Gly Leu Asn Glu Ala Trp Ala Asp 1765 1770 1775 Leu Leu Glu Leu Ile Asp Thr Arg Thr Gln Ile Leu Ala Ala Ser Tyr 1780 1785 1790 Glu Leu His Lys Phe Tyr His Asp Ala Lys Glu Ile Phe Gly Arg Ile 1795 1800 1805 Gln Asp Lys His Lys Lys Leu Pro Glu Glu Leu Gly Arg Asp Gln Asn 1810 1815 1820 Thr Val Glu Thr Leu Gln Arg Met His Thr Thr Phe Glu His Asp Ile 1825 1830 1835 1840 Gln Ala Leu Gly Thr Gln Val Arg Gln Leu Gln Glu Asp Ala Ala Arg 1845 1850 1855 Leu Gln Ala Ala Tyr Ala Gly Asp Lys Ala Asp Asp Ile Gln Lys Arg 1860 1865 1870 Glu Asn Glu Val Leu Glu Ala Trp Lys Ser Leu Leu Asp Ala Cys Glu 1875 1880 1885 Gly Arg Arg Val Arg Leu Val Asp Thr Gly Asp Lys Phe Arg Phe Phe 1890 1895 1900 Ser Met Val Arg Asp Leu Met Leu Trp Met Glu Asp Val Ile Arg Gln 1905 1910 1915 1920 Ile Glu Ala Gln Glu Lys Pro Arg Asp Val Ser Ser Val Glu Leu Leu 1925 1930 1935 Met Asn Asn His Gln Gly Ile Lys Ala Glu Ile Asp Ala Arg Asn Asp 1940 1945 1950 Ser Phe Thr Ala Cys Ile Glu Leu Gly Lys Ser Leu Leu Ala Arg Lys 1955 1960 1965 His Tyr Ala Ser Glu Glu Ile Lys Glu Lys Leu Leu Gln Leu Thr Glu 1970 1975 1980 Lys Arg Lys Glu Met Ile Asp Lys Trp Glu Asp Arg Trp Glu Trp Leu 1985 1990 1995 2000 Arg Leu Ile Leu Glu Val His Gln Phe Ser Arg Asp Ala Ser Val Ala 2005 2010 2015 Glu Ala Trp Leu Leu Gly Gln Glu Pro Tyr Leu Ser Ser Arg Glu Ile 2020 2025 2030 Gly Gln Ser Val Asp Glu Val Glu Lys Leu Ile Lys Arg His Glu Ala 2035 2040 2045 Phe Glu Lys Ser Ala Ala Thr Trp Asp Glu Arg Phe Ser Ala Leu Glu 2050 2055 2060 Arg Leu Thr Thr Leu Glu Leu Leu Glu Val Arg Arg Gln Gln Glu Glu 2065 2070 2075 2080 Glu Glu Arg Lys Arg Arg Pro Pro Ser Pro Asp Pro Asn Thr Lys Val 2085 2090 2095 Ser Glu Glu Ala Glu Ser Gln Gln Trp Asp Thr Ser Lys Gly Asp Gln 2100 2105 2110 Val Ser Gln Asn Gly Leu Pro Ala Glu Gln Gly Ser Pro Arg Val Ser 2115 2120 2125 Tyr Arg Ser Gln Thr Tyr Gln Asn Tyr Lys Asn Phe Asn Ser Arg Arg 2130 2135 2140 Thr Ala Ser Asp His Ser Trp Ser Gly Met 2145 2150 5 8176 DNA Mus musculus 5 cctgcgtcct tcctcctttt cctccttccc tcctccctcc cgggtaattt atttctagct 60 tccaggcaag ggccacacaa ggaaggaaat ccacagggga ttagatgccg gggtggtaac 120 tccaccaggc taggttggac tctgcagcca acttcctatc agatcaccct gcacctattt 180 ccgacccgac cggaatgcga ctggcttgag gtccagccct ttcgcctggg cgggagcaga 240 gccgcggaag ctgcttggag ttggatgggg gtaggaaggg gctggagcgg gaatcctacg 300 atgcaactgg cctgggccta aggttgggca taatggagtt gcagaggaca tccagcgttt 360 cagggccgct gtcgccggcc tacaccgggc aggtgcctta caactacaac caactggagg 420 gaagattcaa acagctccaa gatgagcgtg aagctgtaca gaagaagacc ttcaccaagt 480 gggtcaattc ccaccttgca agagtgtcct gccgaatcac agacctgtac acggaccttc 540 gagatggacg gatgctcatc aagctactgg aggtcctctc tggagagagg ctgcctaaac 600 ccactaaggg acggatgcgg atccactgtc tggagaatgt cgacaaggct cttcaattcc 660 tgaaagagca gagagtccat cttgagaaca tgggctccca tgacattgtg gatggaaacc 720 accggctgac cctcggcctc atctggacaa ttattctgcg cttccagatc caggatatta 780 gtgtggagac tgaagataac aaagagaaaa agtctgctaa ggatgcattg ctgctgtggt 840 gccagatgaa gacagctggg taccccaatg tcaacattca caatttcacc actagctgga 900 gggatggcat ggccttcaat gcactgatac ataaacatcg gcctgacctg atagattttg 960 ataaactgaa gaaatctaat gcacactaca atctgcagaa tgcatttaac ctggcagagc 1020 agcaccttgg cctcactaaa ctgttagacc ctgaagatat cagtgtggac caccctgatg 1080 agaagtctat catcacatac gtggtgactt actaccacta cttctccaag atgaaggcct 1140 tggctgtcga aggaaagcgc attggaaagg tgcttgataa tgctatagaa acagagaaaa 1200 tgattgagaa gtacgagaca cttgcttctg accttctgga gtggattgaa caaaccatca 1260 tcatcctaaa caaccgcaaa tttgctaatt cactggttgg ggtccaacag cagctccaag 1320 cattcaacac gtaccgcaca gtggagaaac cacctaagtt tactgagaag gggaatttgg 1380 aggtgctcct tttcgcgatt cagagcaaga tgcgagcgaa taatcagaag gtctacatgc 1440 cccgcgaggg gaagctcatc tctgacatca acaaggcctg ggaaagactg gaaaaagcag 1500 aacatgagag agaactggct ctgcggaatg agctcatacg gcaggaaaaa ctggaacaac 1560 tcgcccgaag atttgatcgc aaggcagcta tgagggagac atggctgagt gaaaaccagc 1620 gtcttgtgtc tcaggacaac tttggatttg accttcccgc tgttgaggct gctaccaaaa 1680 aacacgaggc cattgagaca gacatcgctg catatgaaga acgagttcag gccgtggtgg 1740 ctgtggccag ggaacttgaa gccgagaact accatgacat caagcgcatc acagcgagga 1800 aggacaatgt catccggctc tgggaatact tgctggaact gctcagggcc aggaggcagc 1860 gtcttgagat gaacctggga ttgcaaaaga tattccagga aatgctttat attatggact 1920 ggatggatga aatgaaggtg ctattgctgt ctcaagacta tggcaaacac ttacttggtg 1980 ttgaagacct gttacagaag catgccctgg ttgaagcaga cattgcaatc caagcagagc 2040 gtgtaagagg tgtgaatgcc tctgcccaga agtttgcaac agatggggaa ggctacaagc 2100 catgtgaccc ccaggtaatt cgagaccgtg ttgcccacat ggagttctgc tatcaagagc 2160 tttgtcagct ggctgccgag cgtagggctc gcctggaaga gtcccgtcgc ctctggaagt 2220 tcttctggga gatggcagaa gaggaaggct ggatacgaga gaaggaaaag atcctgtcct 2280 ctgatgatta cgggaaagac ttgaccagtg tcatgcgcct gctgagcaag caccgggcat 2340 ttgaggatga gatgagtggc cgtagtggcc attttgagca ggccattaaa gaaggtgaag 2400 acatgattgc agaggaacac tttggatcgg aaaagatccg tgagagaatc atttatatcc 2460 gggagcagtg ggccaacctg gaacagctct cagccattag gaagaagcgc ctagaggaag 2520 cctcattact gcaccagttc caggctgatg ctgatgatat tgatgcttgg atgttagata 2580 tactcaagat tgtctccagc aatgatgtgg gccatgatga gtactccacg cagtctctgg 2640 tcaagaagca taaagatgta gcagaagaga tcaccaactg caggcccact attgacacac 2700 tgcatgagca agccagtgcc cttccacaag cacatgcaga gtctccagat gtgaagggcc 2760 ggctggcagg aattgaggag cgctgcaagg agatggcaga gttaacacgg ctaaggaagc 2820 aggctctgca ggacaccctg gccctgtaca agatgttcag tgaggctgat gcctgtgagc 2880 tctggattga cgagaaggag cagtggctca acaacatgca gatcccagag aagctggagg 2940 acctggaagt catccagcac agatttgaga gcctagaacc agaaatgaac aaccaggctt 3000 cccgggttgc tgtggtgaac cagattgcac ggcagctgat gcacaatggc caccccagtg 3060 aaaaggaaat cagagctcag caagacaaac tcaacacgag gtggagtcag ttcagagaac 3120 tggtggacag gaaaaaggat gctcttctgt ctgccctgag catccagaac taccacctcg 3180 agtgcaatga aaccaaatcc tgcatccggg agaagaccaa ggtcatcgag tctacccaag 3240 accttggcaa tgacctggca ggtgtcatgg ccctgcagtg caagctgact ggcatggaac 3300 gagacttggt agccattgag gcgaagctga gtgacctgca gaaagaagct gagaagctgg 3360 agtccgagca ccctgaccag gctcaagcta tcctgtctcg gctggccgag atcagtgatg 3420 tgtgggagga aatgaagaca accctgaaga accgagaggc ctccctggga gaggccagca 3480 agctgcagca gtttctgcgg gacttggacg acttccagtc ttggctctcc aggacccaga 3540 ctgctatcgc ctcagaggac atgcccaata ccctcactga ggcagagaag cttctcacac 3600 agcacgagaa tatcaaaaat gagatcgaca attatgagga agactaccag aagatgcggg 3660 acatgggcga gatggtcacc caggggcaga ctgatgccca gtatatgttt ctgcggcagc 3720 ggctgcaggc cttagacact ggctggaatg agctccacaa aatgtgggag aacaggcaaa 3780 acctcctctc ccagtcccat gcctaccagc agttccttag ggacaccaaa caagctgaag 3840 cttttcttaa taaccaggag tatgttttgg ctcatactga aatgcccacc accctggaag 3900 gagctgaagc agccattaaa aagcaggagg acttcatgac caccatggat gccaacgagg 3960 agaagatcaa tgctgttgtg gagactggcc gaagactggt gagcgatggg aacatcaact 4020 ccgaccgcat ccaggagaag gtggactcta ttgacgacag acacaggaag aatcgagaag 4080 cagccagtga acttctgatg aggttaaagg acaaccgtga tctacagaag ttcctgcaag 4140 attgtcaaga gctgtccctc tggatcaatg aaaagatgct tacagctcaa gacatgtctt 4200 atgatgaagc cagaaatctg cacagtaaat ggttaaagca tcaagcattt atggcggaac 4260 ttgcatccaa caaagaatgg cttgacaaaa ttgagaagga aggaatgcag cttatttcag 4320 aaaagccaga aacagaagct gtggtaaagg aaaaactcac tggtttacat aaaatgtggg 4380 aagtccttga atccacaacc cagaccaagg cccagcggct ctttgatgca aataaggctg 4440 agcttttcac acaaagctgc gcagatcttg acaaatggct acatggcctg gagagccaga 4500 ttcaatctga cgactatggc aaagacctta ccagtgtcaa tattcttctg aaaaagcaac 4560 agatgctgga gaatcagatg gaagttcgga agaaagagat cgaggaactg cagagccaag 4620 cccaggcgct gagtcaggag gggaagagca cagatgaggt ggacagcaaa cgccttactg 4680 tgcagaccaa gttcatggag cttctggagc ccttgagtga gaggaagcat aacctgttag 4740 cttccaagga gatccatcag ttcaacaggg atgtggagga cgaaatccta tgggttggcg 4800 agaggatgcc tttggcaact tccacagatc atggccataa ccttcaaact gtgcagctgt 4860 taataaagaa aaaccagacc ctccagaaag aaatccaggg acaccagcct cgtattgatg 4920 acatctttga gaggagtcaa aacatcatca cagatagcag cagcctcaat gccgaggcta 4980 tcaggcagag gctcgctgac ctgaagcagc tgtgggggct cctcattgag gaaactgaga 5040 aacgccatag acggctggag gaggcacaca aggcgcagca gtactacttt gatgcagctg 5100 aagccgaggc atggatgagt gaacaggagt tgtacatgat gtctgaggaa aaggccaagg 5160 atgagcagag tgctgtctct atgttgaaaa agcaccagat tttagagcaa gctgttgagg 5220 actatgcaga gacagtacac cagctctcca agactagccg ggcgctggtg gctgacagcc 5280 atcccgaaag tgagcgtatt agcatgcggc agtcaaaggt cgacaagctg tatgctggcc 5340 tgaaggacct tgctgaggag aggagaggaa aacttgatga gaggcacagg ctgttccagc 5400 tcaacagaga ggtggatgac ctggaacagt ggatcgctga gagggaagtg gtcgcaggct 5460 cccatgagtt gggacaggac tatgagcatg tcacgatgtt acaagaacgg ttccgagaat 5520 ttgctcgaga cacaggaaac attgggcagg agcgtgtgga tacagttaat aacatggcag 5580 atgaactcat caactctgga cattcagatg ctgccaccat tgctgagtgg aaagatggtc 5640 tcaatgaagc ctgggctgac ctcctggagc tcattgacac aagaacacag attcttgctg 5700 cctcatatga acttcataag ttttaccatg atgccaagga gatctttggc cgaatccagg 5760 acaaacacaa gaaactccct gaggagcttg gaagagatca aaacactgtg gaaactttac 5820 agagaatgca caccaccttt gagcacgaca tccaagctct gggcactcag gtgaggcagc 5880 tgcaggagga tgcagctcgc ctccaggcag cctatgcagg ggacaaggct gatgacatcc 5940 agaagcgtga gaatgaggtc ctggaagcct ggaagtccct gctggatgct tgtgagggtc 6000 gcagggtgcg gctggtagac acaggagaca agttccgctt cttcagcatg gtgcgtgacc 6060 tcatgctctg gatggaagat gtcatccggc agatcgaggc ccaggagaaa ccacgggatg 6120 tgtcatctgt tgaactgtta atgaataatc atcaaggtat caaagctgaa attgatgctc 6180 gtaatgacag ctttacagcc tgcattgagc ttgggaaatc cctgctggca cggaaacact 6240 atgcttctga ggagatcaag gaaaagttac tgcagctgac agagaaaaga aaagaaatga 6300 ttgacaagtg ggaagaccgg tgggagtggt taagactgat tttggaggtc catcagttct 6360 caagggatgc cagtgtggca gaggcttggc tgcttggaca ggaaccatac ctatccagcc 6420 gtgaaattgg ccagagtgta gacgaagtgg agaagcttat taagcgccat gaggcgtttg 6480 aaaagtctgc agcgacctgg gatgagagat tctctgctct ggaaaggctg acaacgttgg 6540 agctactgga agtgcgcaga cagcaagagg aagaagaaag aaagaggcgg ccaccttctc 6600 cggacccaaa cacgaaggtt tcagaggagg ctgagtccca gcaatgggat acttcaaaag 6660 gagaccaagt ttcccagaat ggtttgccgg ctgagcaggg atctccacgg gttagttacc 6720 gctctcaaac gtaccaaaac tacaaaaact ttaatagcag acggacagcc agtgaccatt 6780 catggtctgg aatgtgaagt tcactaccat ttgtcaagaa ccactctgtc cacatccttt 6840 gaccttttgg cttccacgtc acccagagtg ttaaaatttt tacttaattc atagctgtcc 6900 ttgatttcat atttgtttgc atttaattta tgtttctttg gatcctcatt gcctcaaagc 6960 agcatactta atttttgttt atttattgtg agctttttac tttaagattt tacatgagta 7020 atcaaaatta aattatagca taatgaaatt agactcttaa caggtacggc acacacaagt 7080 taatagtact ctgctatagg tgctatgtta cttacaagta ttattaacct attggcttcc 7140 attgtatagt agttagtaac tatgaaaact ggtttgtaag gaaggaaacg tttactacta 7200 aggttaggcc tgcagttgct ctggaacatt ccatggagaa tgcattcatc aaacggcccg 7260 aaagaagcta cattttgttg ggaagctgga taagttttag gtgcaggacc ccaaatgttc 7320 tgagaccttt ggggccattt attactttgt acaagcccaa taatcctctc ttttctgcca 7380 agtcctcaac ccagaaatgt aggcttctgt gcaccacacg gcacagccca ctgattgctg 7440 ccaccggctc tgtcttggtc agtgttacca ctgccagcac tcaggctgtg gcagatgcca 7500 gcagctctta ccatcagtca gagtcttcag ggtgtcaagc tgttttcatt ttttaggcaa 7560 atagaacaaa agccattttg gttcatcctg atcacttgaa tgatagactc aatgccctgt 7620 gcctggcagg gagcgcttgc agaggtgtcc tagccttaga gggctacttc agtgtctcta 7680 ctgacagaaa ctcctgtatc tcaaatggat ctcgaagttc tctagtaagg agtcctaagg 7740 atgacatgta ttgggccact agcagggatt gaaaacattt taaaagaaat cctttttctt 7800 aggagtaaaa gctggtaaaa ggggtgactt cctggttctg atcaaaacca gaccaaaccc 7860 tcatttcagc aaagccttgc aagacactcc cttgctcatt tgccatattt agatgtctta 7920 gtggagtcag agccctgttt ggtatgtgtt tttcatgcta agtctaaatt gtcttttcat 7980 ttcatgatgc attttttctc ttttgtcagg ataacatcat atagcatctt gtttgttttt 8040 cctaatctct atgaacatat ctatctacct gtaaccgtag ataggtatct agatagatac 8100 caagctttta agctctgggc cactatgcat cattattggg tctctgcctt aaaacacatc 8160 caaatttata ttaaaa 8176 6 1312 DNA Mus musculus CDS (402)..(1061) 6 gcgctgctct gtgagctgga gcacagcgtg cttagagttg gccatattta aaatattttc 60 caataggatc ctgcgtcctt cctccttttc ctccttccct cctccctccc gggtaattta 120 tttctagctt ccaggcaagg gccacacaag gaaggaaatc cacaggggat tagatgccgg 180 ggtggtaact ccaccaggct aggttggact ctgcagccaa cttcctatca gatcaccctg 240 cacctatttc cgacccgacc ggaatgcgac tggcttgagg tccagccctt tcgcctgggc 300 gggagcagag ccgcggaagc tgcttggagt tggatggggg taggaagggg ctggagcggg 360 aatcctacgg tgcaactggc ctgggcctaa ggttgggcat a atg gag ttg cag agg 416 Met Glu Leu Gln Arg 1 5 aca tcc agc att tca ggg ccg ctg tcg ccg gcc tac acc ggg cag gtg 464 Thr Ser Ser Ile Ser Gly Pro Leu Ser Pro Ala Tyr Thr Gly Gln Val 10 15 20 cct tac aac tac aac caa ctg gaa gga aga ttc aaa cag ctc caa gat 512 Pro Tyr Asn Tyr Asn Gln Leu Glu Gly Arg Phe Lys Gln Leu Gln Asp 25 30 35 gag cgt gaa gct gta cag aag aag acc ttc acc aag tgg gtc aat tcc 560 Glu Arg Glu Ala Val Gln Lys Lys Thr Phe Thr Lys Trp Val Asn Ser 40 45 50 cac ctt gcg aga gtg tcc tgc cga atc aca gac ctg tac acg gac ctt 608 His Leu Ala Arg Val Ser Cys Arg Ile Thr Asp Leu Tyr Thr Asp Leu 55 60 65 cga gat gga cgg atg ctc atc aag cta ctg gag gtc ctc tct gga gag 656 Arg Asp Gly Arg Met Leu Ile Lys Leu Leu Glu Val Leu Ser Gly Glu 70 75 80 85 agg ctg cct aaa ccc act aag gga cgg atg cgg atc cac tgt ctg gag 704 Arg Leu Pro Lys Pro Thr Lys Gly Arg Met Arg Ile His Cys Leu Glu 90 95 100 aat gtc gac aag gct ctt caa ttc ctg aaa gag cag aga gtc cat ctt 752 Asn Val Asp Lys Ala Leu Gln Phe Leu Lys Glu Gln Arg Val His Leu 105 110 115 gag aac atg ggc tcc cat gac att gtg gat gga aac cac cgg ctg aca 800 Glu Asn Met Gly Ser His Asp Ile Val Asp Gly Asn His Arg Leu Thr 120 125 130 acg ttg gag cta ctg gaa gtg cgc aga cag caa gag gaa gaa gaa aga 848 Thr Leu Glu Leu Leu Glu Val Arg Arg Gln Gln Glu Glu Glu Glu Arg 135 140 145 aag agg cgg cca cct tct ccg gac cca aac acg aag gtt tca gag gag 896 Lys Arg Arg Pro Pro Ser Pro Asp Pro Asn Thr Lys Val Ser Glu Glu 150 155 160 165 gct gag tcc cag caa tgg gat act tca aaa gga gac caa gtt tcc cag 944 Ala Glu Ser Gln Gln Trp Asp Thr Ser Lys Gly Asp Gln Val Ser Gln 170 175 180 aat ggt ttg ccg gct gag cag gga tct cca cgg gtt agt tac cgc tct 992 Asn Gly Leu Pro Ala Glu Gln Gly Ser Pro Arg Val Ser Tyr Arg Ser 185 190 195 caa acg tac caa aac tac aaa aac ttt aat agc aga cgg aca gcc agt 1040 Gln Thr Tyr Gln Asn Tyr Lys Asn Phe Asn Ser Arg Arg Thr Ala Ser 200 205 210 gac cat tca tgg tct gga atg tgaagttcac taccatttgt caagaaccac 1091 Asp His Ser Trp Ser Gly Met 215 220 tctgtccaca tcctttgacc ttttggcttc cacgtcaccc agagtgttaa aatttttact 1151 taattcatag ctgtccttga tttcatattt gtttgcattt aatttatgtt tctttggatc 1211 ctcattgcct caaagcagca tacttaattt ttgtttattt attgtgagct ttttacttta 1271 agattttaca tgagtaatca aaattaaatt atagcataat g 1312 7 220 PRT Mus musculus 7 Met Glu Leu Gln Arg Thr Ser Ser Ile Ser Gly Pro Leu Ser Pro Ala 1 5 10 15 Tyr Thr Gly Gln Val Pro Tyr Asn Tyr Asn Gln Leu Glu Gly Arg Phe 20 25 30 Lys Gln Leu Gln Asp Glu Arg Glu Ala Val Gln Lys Lys Thr Phe Thr 35 40 45 Lys Trp Val Asn Ser His Leu Ala Arg Val Ser Cys Arg Ile Thr Asp 50 55 60 Leu Tyr Thr Asp Leu Arg Asp Gly Arg Met Leu Ile Lys Leu Leu Glu 65 70 75 80 Val Leu Ser Gly Glu Arg Leu Pro Lys Pro Thr Lys Gly Arg Met Arg 85 90 95 Ile His Cys Leu Glu Asn Val Asp Lys Ala Leu Gln Phe Leu Lys Glu 100 105 110 Gln Arg Val His Leu Glu Asn Met Gly Ser His Asp Ile Val Asp Gly 115 120 125 Asn His Arg Leu Thr Thr Leu Glu Leu Leu Glu Val Arg Arg Gln Gln 130 135 140 Glu Glu Glu Glu Arg Lys Arg Arg Pro Pro Ser Pro Asp Pro Asn Thr 145 150 155 160 Lys Val Ser Glu Glu Ala Glu Ser Gln Gln Trp Asp Thr Ser Lys Gly 165 170 175 Asp Gln Val Ser Gln Asn Gly Leu Pro Ala Glu Gln Gly Ser Pro Arg 180 185 190 Val Ser Tyr Arg Ser Gln Thr Tyr Gln Asn Tyr Lys Asn Phe Asn Ser 195 200 205 Arg Arg Thr Ala Ser Asp His Ser Trp Ser Gly Met 210 215 220 8 1964 DNA Mus musculus CDS (322)..(1509) 8 ttggaacagt tacttcagtg gaggcagcag aaatgaggct agtccagact cacaggaata 60 gggttccatt ctcaagaaga tgatttaaag taattatcct ttacgcatag ttatcatcac 120 cacaaaaaaa gattccaacc ttttccacag aactattatg atttattttt atatgaatgt 180 atgtatttat tattatatga actcctataa tgatcacctt tacatattca cattttctta 240 ataattagtt tagccgcgtc cggaggtccg acagctctgc agctccgagc gcgcgactag 300 ccagaaagtt tcaggccatc c atg agc cac cag gaa agg att gcc agc cag 351 Met Ser His Gln Glu Arg Ile Ala Ser Gln 1 5 10 agg agg aca aca gcc gaa gtc cca atg cac aga tca act gcc aat caa 399 Arg Arg Thr Thr Ala Glu Val Pro Met His Arg Ser Thr Ala Asn Gln 15 20 25 agc aag agg agc cgg tca cca ttt gcc agc aca cgt cgt cgc tgg gat 447 Ser Lys Arg Ser Arg Ser Pro Phe Ala Ser Thr Arg Arg Arg Trp Asp 30 35 40 gac agc gag agc tcg gga gcc agc ctg gct gtt gag agt gag gat tat 495 Asp Ser Glu Ser Ser Gly Ala Ser Leu Ala Val Glu Ser Glu Asp Tyr 45 50 55 tcc agg tgg cgg gat gct gcc gat gct gag gag gct cat gcc gag ggc 543 Ser Arg Trp Arg Asp Ala Ala Asp Ala Glu Glu Ala His Ala Glu Gly 60 65 70 cta gcc aga aga ggc cga ggt gag gct gcc agc agc tca gag cca agg 591 Leu Ala Arg Arg Gly Arg Gly Glu Ala Ala Ser Ser Ser Glu Pro Arg 75 80 85 90 tat gct gaa gac cag gat gcc agg agt gaa caa gcg aag gca gac aaa 639 Tyr Ala Glu Asp Gln Asp Ala Arg Ser Glu Gln Ala Lys Ala Asp Lys 95 100 105 gtg cca aga cgg cgg cga acc atg gca gac cct gac ttc tgg gca tac 687 Val Pro Arg Arg Arg Arg Thr Met Ala Asp Pro Asp Phe Trp Ala Tyr 110 115 120 acc gac gat tac tac cga tac tac gag gaa gat tct gac agc gac aaa 735 Thr Asp Asp Tyr Tyr Arg Tyr Tyr Glu Glu Asp Ser Asp Ser Asp Lys 125 130 135 gag tgg atg gct gcc ctg cgc agg aag tac cga agc cga gag caa ccc 783 Glu Trp Met Ala Ala Leu Arg Arg Lys Tyr Arg Ser Arg Glu Gln Pro 140 145 150 cag tcc tcc agc gga gaa agc tgg gag ctt ctg cca gga aag gaa gaa 831 Gln Ser Ser Ser Gly Glu Ser Trp Glu Leu Leu Pro Gly Lys Glu Glu 155 160 165 170 ctg gaa cgt cag caa gcc gga gct ggg agc ctc gcc agt gct ggc agc 879 Leu Glu Arg Gln Gln Ala Gly Ala Gly Ser Leu Ala Ser Ala Gly Ser 175 180 185 aat ggc agt ggt tat cct gaa gaa gta caa gac cca tct ctt cag gag 927 Asn Gly Ser Gly Tyr Pro Glu Glu Val Gln Asp Pro Ser Leu Gln Glu 190 195 200 gaa gaa cag gcc tct ctg gaa gaa gga gaa atc cct tgg ctt cgc tac 975 Glu Glu Gln Ala Ser Leu Glu Glu Gly Glu Ile Pro Trp Leu Arg Tyr 205 210 215 aat gag aat gaa agc agc agc gag ggt gat aat gag tct acc cat gag 1023 Asn Glu Asn Glu Ser Ser Ser Glu Gly Asp Asn Glu Ser Thr His Glu 220 225 230 ctc ata cag cct ggg atg ttc atg ctg gat gga aac aac aac ctg gaa 1071 Leu Ile Gln Pro Gly Met Phe Met Leu Asp Gly Asn Asn Asn Leu Glu 235 240 245 250 gat gac tcc agc gtg agc gaa gac ctc gaa gtg gac tgg agc ctg ttt 1119 Asp Asp Ser Ser Val Ser Glu Asp Leu Glu Val Asp Trp Ser Leu Phe 255 260 265 gat ggg ttt gcc gat ggc ttg gga gtg gcc gaa gcc atc tcc tac gtg 1167 Asp Gly Phe Ala Asp Gly Leu Gly Val Ala Glu Ala Ile Ser Tyr Val 270 275 280 gat cct cag ttc ctc acc tac atg gct ctg gaa gag cgt ctg gcc cag 1215 Asp Pro Gln Phe Leu Thr Tyr Met Ala Leu Glu Glu Arg Leu Ala Gln 285 290 295 gca atg gag acg gcc ctg gca cac ttg gag tct ctc gcc gtt gat gtc 1263 Ala Met Glu Thr Ala Leu Ala His Leu Glu Ser Leu Ala Val Asp Val 300 305 310 gaa gtg gcc aac cca cca gca agc aag gag agc att gat gcc ctt cct 1311 Glu Val Ala Asn Pro Pro Ala Ser Lys Glu Ser Ile Asp Ala Leu Pro 315 320 325 330 gag atc ctg gtc acc gaa gat cat ggt gca gtg ggc cag gaa atg tgc 1359 Glu Ile Leu Val Thr Glu Asp His Gly Ala Val Gly Gln Glu Met Cys 335 340 345 tgt cct atc tgc tgc agc gaa tat gtg aag ggg gag gtg gca act gag 1407 Cys Pro Ile Cys Cys Ser Glu Tyr Val Lys Gly Glu Val Ala Thr Glu 350 355 360 cta cca tgc cac cac tat ttc cac aag ccc tgc gtg tcc atc tgg ctt 1455 Leu Pro Cys His His Tyr Phe His Lys Pro Cys Val Ser Ile Trp Leu 365 370 375 cag aag tct ggc acc tgc cca gtg tgc cgc tgc atg ttc cct ccc ccg 1503 Gln Lys Ser Gly Thr Cys Pro Val Cys Arg Cys Met Phe Pro Pro Pro 380 385 390 ctc taa aagccaaggc tcgtcgtaac agtcagcctg gttacattcc ctgtccgaaa 1559 Leu 395 cccacaatac tacaggagcc cttgttctaa acttacaatg aaaccagtca gtcaattaga 1619 ctaaagttgt tgattccttg tgattatttc catgtgaaaa tggttgtgta caatgacatt 1679 taaaaaaaat catcctctcg tttagaaggt agaaaggggg aaaggaaact ttctaaatgc 1739 tgcttgagat tgcagtaaga acatacattt tctaacctga aagttgaaac aaatcccact 1799 tgttctgtag actgtgtctc tcttacctgt tgctgtcagg gttacctatc tgctaaacta 1859 tgtcggaaag acaaaattac ttttgttgca tgtcatgggt taatgttcct gtatttgcag 1919 tggtgtaaaa gcttattaaa gttcttcttt tgctttgacc ccgaa 1964 9 395 PRT Mus musculus 9 Met Ser His Gln Glu Arg Ile Ala Ser Gln Arg Arg Thr Thr Ala Glu 1 5 10 15 Val Pro Met His Arg Ser Thr Ala Asn Gln Ser Lys Arg Ser Arg Ser 20 25 30 Pro Phe Ala Ser Thr Arg Arg Arg Trp Asp Asp Ser Glu Ser Ser Gly 35 40 45 Ala Ser Leu Ala Val Glu Ser Glu Asp Tyr Ser Arg Trp Arg Asp Ala 50 55 60 Ala Asp Ala Glu Glu Ala His Ala Glu Gly Leu Ala Arg Arg Gly Arg 65 70 75 80 Gly Glu Ala Ala Ser Ser Ser Glu Pro Arg Tyr Ala Glu Asp Gln Asp 85 90 95 Ala Arg Ser Glu Gln Ala Lys Ala Asp Lys Val Pro Arg Arg Arg Arg 100 105 110 Thr Met Ala Asp Pro Asp Phe Trp Ala Tyr Thr Asp Asp Tyr Tyr Arg 115 120 125 Tyr Tyr Glu Glu Asp Ser Asp Ser Asp Lys Glu Trp Met Ala Ala Leu 130 135 140 Arg Arg Lys Tyr Arg Ser Arg Glu Gln Pro Gln Ser Ser Ser Gly Glu 145 150 155 160 Ser Trp Glu Leu Leu Pro Gly Lys Glu Glu Leu Glu Arg Gln Gln Ala 165 170 175 Gly Ala Gly Ser Leu Ala Ser Ala Gly Ser Asn Gly Ser Gly Tyr Pro 180 185 190 Glu Glu Val Gln Asp Pro Ser Leu Gln Glu Glu Glu Gln Ala Ser Leu 195 200 205 Glu Glu Gly Glu Ile Pro Trp Leu Arg Tyr Asn Glu Asn Glu Ser Ser 210 215 220 Ser Glu Gly Asp Asn Glu Ser Thr His Glu Leu Ile Gln Pro Gly Met 225 230 235 240 Phe Met Leu Asp Gly Asn Asn Asn Leu Glu Asp Asp Ser Ser Val Ser 245 250 255 Glu Asp Leu Glu Val Asp Trp Ser Leu Phe Asp Gly Phe Ala Asp Gly 260 265 270 Leu Gly Val Ala Glu Ala Ile Ser Tyr Val Asp Pro Gln Phe Leu Thr 275 280 285 Tyr Met Ala Leu Glu Glu Arg Leu Ala Gln Ala Met Glu Thr Ala Leu 290 295 300 Ala His Leu Glu Ser Leu Ala Val Asp Val Glu Val Ala Asn Pro Pro 305 310 315 320 Ala Ser Lys Glu Ser Ile Asp Ala Leu Pro Glu Ile Leu Val Thr Glu 325 330 335 Asp His Gly Ala Val Gly Gln Glu Met Cys Cys Pro Ile Cys Cys Ser 340 345 350 Glu Tyr Val Lys Gly Glu Val Ala Thr Glu Leu Pro Cys His His Tyr 355 360 365 Phe His Lys Pro Cys Val Ser Ile Trp Leu Gln Lys Ser Gly Thr Cys 370 375 380 Pro Val Cys Arg Cys Met Phe Pro Pro Pro Leu 385 390 395 10 2992 DNA Mus musculus 10 gggcaactga aggcagatga agagccctgc ccctgcccac atgtggaacc ttgtgctgtt 60 cttgccttca ctgttggctg tgcttccgac cactactgcc gagaagaatg gcatcgatat 120 ctacagcctc acggtggact cccgggtctc ttcccgattt gcccatactg ttgtcaccag 180 ccgggtggtc aacagagccg atgctgttca agaagcgacc ttccaagtag agctacccag 240 gaaagccttc atcaccaact tctccatgat catcgatggc gtgacctacc caggggttgt 300 caaagagaag gccgaagccc agaaacaata cagtgccgcc gtgggcaggg gagagagtgc 360 tggcatcgtc aagaccactg ggaggcagac agagaagttt gaagtgtcag tcaacgtggc 420 ccctggttcc aagattacct tcgaactcat ataccaggaa ctgctccaaa ggcgactggg 480 aatgtatgag ctactcctca aagtgaggcc tcagcagctg gtgaagcacc ttcagatgga 540 catctacatc tttgagcctc agggtattag catcctggag acagagagca ccctcatgac 600 cccggagctg gcaaatgccc ttaccacttc acagaacaag accaaggctc atatccggtt 660 caagccgacg ctctcccagc aacagaagtc tcagagtgag caggacacgg tgctgaatgg 720 ggacttcatc gtccgctatg atgtcaaccg gtctgactct gggggctcca ttcagattga 780 ggaaggctac tttgtgcacc actttgctcc agagaacctt cctacaatgt ccaagaatgt 840 gatctttgtc attgataaaa gcggatctat gtcaggcaag aaaatccagc agacccgaga 900 agccctagtc aagatcttga aagacctcag cccccaagac cagttcaacc tcattgagtt 960 cagtggggaa gcaaaccaat ggaagcagtc actggtgcaa gcgacagaag agaatttgaa 1020 caaggctgta aactatgctt ccaggatccg ggctcacgga gggaccaaca tcaataatgc 1080 agtgctgttg gctgtggagc tgctggacag aagcaaccaa gctgagctac tgccctcgaa 1140 gagcgtctcc cttatcatcc tgctcacgga cggtgacccc actgtgggag aaaccaaccc 1200 cacgattatc cagaacaacg tgcgggaagc catcaatggg cagtatagcc tcttctgcct 1260 ggggttcggc tttgatgtga actatccttt cctggagaag atggcactgg acaatggtgg 1320 cctggccagg cgcatctatg aggattcaga ctctgcactg cagcttcagg atttctacca 1380 cgaagtagcc aatccactgc tctcatcagt ggccttcgaa taccccagtg atgctgtgga 1440 ggaagtcact cggtacaagt tccaacacca ctttaagggc tcagagatgg tggtggctgg 1500 gaagctccag gaccagggtc ctgatgtcct cttagccaaa gtcagtgggc agatgcacat 1560 gcagaacatc actttccaaa cggaggccag cgtagcccaa caagagaagg agtttaagag 1620 ccccaagtac atctttcaca actttatgga gagactgtgg gcactgctga ctatacagca 1680 acagctggag cagaggattt cagcgtcagg tgccgaatta gaggccctcg aggcccaagt 1740 tctgaacttg tcactcaagt acaattttgt cacccctctc acgcacatgg tggtcaccaa 1800 acctgaaggt caagaacaat tccaagttgc tgagaagcct gtggaagtcg gtgatggcat 1860 gcagagactc cccttagcag ctcaagccca ccccttcagg cctcctgtca gaggatctaa 1920 actgatgacc gtgctgaaag gaagcaggtc ccagataccc agacgcggtg atgccgttag 1980 ggcatctagg caatacattc ctcccggatt ccccggacct cctggacctc ccggatttcc 2040 tgcaccccct ggacctcctg gatttcctgc accccctgga cctcctcttg cttctggctc 2100 tgacttcagc cttcagcctt cctatgaaag gatgctaagc ctgccctccg ttgcagcaca 2160 atatcctgct gacccacatc tggttgtgac ggaaaaaagt aaagaaagca ccataccaga 2220 ggaatcccca aacccagacc acccccaggt tcctactatt accttgccgc ttccgggatc 2280 cagtgtggac cagctctgtg tggatatctt acattctgag aagcccatga agctgttcgt 2340 agaccccagt cagggtctgg aggtgactgg taagtatgag aatactgggt tctcgtggct 2400 cgaagtgacc atccagaagc ctcacctgca ggtccatgca acccctgaac gactggtggt 2460 gacacgaggc agaaaaaaca ctgaatacaa gtggaagaag acgctgttct ctgtgttacc 2520 tggcttgaag atgaccatga atatgatggg actcctacag ctcagtggcc cagacaaagt 2580 caccatcggc ctcctgtccc tggatgaccc tcagagagga ctaatgctgc ttttgaatga 2640 cacccagcac ttctccaaca acgttaaagg ggagcttggt cagttttacc gggacatcgt 2700 ctgggagcca cccgtcgagc cagataatac aaaacggaca gtcaaagttc aaggagttga 2760 ctacctggct accagagagc tcaagttgag ttaccaagaa gggttcccag gagcagagat 2820 ttcctgctgg acagtggaga tatagaactg ttaggagcgc cgctccctgc catgttgtcc 2880 tcgtacgcag gcagatgaca ccttatgcca acagggacgc ctgtgaggcc gagaccttga 2940 tgggaagagg atgctccctt gttacaaata aagaagggca gtgtgaaccc ga 2992 11 1177 DNA Mus musculus misc_feature n=(a or c or t or g) 11 ggtggccaag agcagttcac ctgctctggg gcaagccttg cttgtgtttt agtgagtcag 60 ggcctcccca ggcagtaaga tgttgagtgt ggaggcccag gccgctgacc tgcagccctg 120 tcccccacag gcaggctgca tgctcttccc ccacatttct ccttgcgagg tgcgcgtgct 180 catgctcctg tactcgtcta agaagaagat cttcatgggc ctcatcccct acgaccagag 240 cggnttcgtc aacgccatac gacaggtcat caccacccgc aaacaggtgt gccagctgag 300 ggtagnctgc tcctgctcct acccttggta gacccactgn ctcccactgg tgtggaatgt 360 ggcatcaagg ctgagtcggc gnctggggag gagctgtgac gangcagtgc catacccaaa 420 tgggctcgag ggaaacntag ctttataggc ttcagagggg cagaactaga gggtggggcc 480 tgggtgtaga ggcagggcag gagtggggtg gcaggtttgg caagaggccc agagtctctg 540 gagggtcaca gtgttgatga catctttctn agaancctgc tactngctta gncagctgtg 600 gtcctctctn ccacctgggg gatacctggc nacaggcngt gggcnncggg ggtgaanact 660 ctggacctgt tnagantgtc aacaacaaat tcttgacatg gagtggtgtc atggagtggn 720 aggaggtgan ctgccgggga ctgtgtggac tgttgnccct aagctgccct cccctgaagt 780 gccttctcgc tctgccccaa aacccagacc tgagcccaac agccggtcca agaggtggct 840 gccatcccac gtctatgtga accaagggga gatcctgtga ttccgggtac ccccgggtgg 900 ccccattgac agtgccgccc ccctggggga ggacttctga ctgatacctc ctgtcttgtg 960 tggcaggaga acagaccagt ggcctcggag gctcttcatg cagctcattc cccagcagtt 1020 gctggtgagg ggtcagggga ttccaggctg ggggtgggcc aaagaccctg tggtgggctg 1080 gttcagaggc ctgcctggct tccccagcaa gctagggttc cataaagaag ccctcggcct 1140 tcccccagac caccctcgtg ccactgttcc ggaattc 1177 12 2998 DNA Mus musculus misc_feature n=(a or c or t or g) 12 ggcacgagct taactgtgct aacttctgtg atgatcatgt gtgatgagta tgtgctctca 60 tttgatttgt gggaaaaaac aaaacaaaaa aatccgaagg acacaaagag gactaatctt 120 aaaccagata tctagtagtc accaaagcca cactttgaat tcgaaaagct tagcactgta 180 gcttagctca tgctatcttt taaagagaga atttaattat ttaatatatg gaaggacatt 240 aggctagtgt gtctggcaca tggtataaac tcaataaatg gtggacgtta tcagtgctac 300 tataatgagt ttaataattt ggtttcatct cctttaatca gaccagtgtt cactactagc 360 tgggtctctg gaataggcac agatatattc atctggagtg tcacacatac tctgtgcgcg 420 aaagagttca gaatagccct tcaataagcc aattactctt gctgtcatcc ttatttctta 480 actttccctt agcgttgctt ttatgtatca aacttttctt ccttatttta cgtaatactt 540 ttaatgacaa ctttctagaa ataagaacta taccctaaaa gattgaaata ttcttagttt 600 tctttatcta catcagaaat tgtttagctg atacaacata cttatattgt ttaaggaatt 660 ctgtttaata ccttggtatt tataattttc ataagtttat ttgtattaat aggaactctt 720 acaaagaatg tatagaaaat aagccccatc atttgtcagt gtgacaattt tcccagtgtt 780 taaattgttt aagctgtttg tacccctata taagctctgt tccttctttg gccctttccc 840 ccttagccta aatctccatt ttgcctgacg atctcttccc tgacaaaatg cctgcttctg 900 cgcactgagt cacagtctac taaaatgcat tccattgtgc ccatgtccct cttaatgtga 960 tgaccccaga catgaccagg gcagagcaca gagggagcat cactttcttt gaccagagca 1020 tctatttcca gcaatgcagc ctaaggtcac attagcattt ttggcagcaa aatacaccct 1080 tggctcatgc tgttatgctg tcaaccaaat cctccatgac tttttcacat gaactcccat 1140 taaataaggc ttcccacatc cggtacgaat atagacagta atgtgcagtc tggtgaagtt 1200 atttacataa gttcctatta aacatcagct aatctatatt tattatttta gaatattgag 1260 acagatttct attcccagct atatagatat ggttttagaa tactttatta ttattttttt 1320 aatgtgtctt ctctgaaccc gataagaaca tagtcccaga caatctttaa gttcagagtc 1380 ttacagtttg tatagagacc tagaggctag ctatatttct ttagacatca acacatcatc 1440 agataggatc cacccaaggg ccttacaaat cctgtatact gaaatgcctt ttcctgacga 1500 tattctggag actgttaagt gaatgcgcag atctgaaccg agccgagcct gtagtgggga 1560 agagctaaag catggcagtt gtcttcatca atgatggagt ctttcattat gttgtctcaa 1620 aagacacatg cttcagccct gggtctcaaa actctcatgc ttcggccctg ggtctcacac 1680 tcctggcttc ccgagtggtc atagctaaga ccttctcaca ctaaatccca ggatgagctc 1740 atgttgatgt tcctgcttgc ttctctgaaa ttggcagttc tcgtgggaaa aaaaatctac 1800 ttatacttgt gtgcttcata aagcaactcg gtagcagggc ttaggggtgc ttcgagtgtg 1860 gcagtgatag agaagaccga taaagcgaaa tctatgatat ctcatacatc attttaatta 1920 tttaaattac ttttgttagt acacaaaagt attttgttag tacaccctgt ttatctatgt 1980 gtatactcta cctttcgcat acactgactt catttctttt tctcctcacc catcctgatg 2040 agctgctctc ctcccagaca agctctggca gttttaaagt cacgtgtgta tcttttaact 2100 ctagcttctg cctattagac aaaacaagat acttgtcttt ctccccatct ccctcctttt 2160 gtttaattct cctccagccc tacatggatc ccccttgacc tcgtgtcata tatctaaatc 2220 tgtataaata aagagatgat ttaatctacg ttctatgtac aaaagagaat ataaatgctc 2280 gtctttctga atctgtctta tttggtttca cacaatatct gctctctttt accgcaaatg 2340 gtatcatctc gttcccttta cacgttgaag aaaatttcat tttgtgtgtg tgtgtgtgtg 2400 tgtgtgtgtg aactatatat ttttacgcta tctggtgagg aacatcaagg ccaagatatg 2460 gatcttggct attgtaaaga gtgtagtaag aaacacaacc gtataatcat ctctgttgca 2520 tgctggcatg ctggctacaa tcctcacctg tgtacccaga gtgagagctg gaccacatgg 2580 taatgcaacc tgtagttatt atttaatgtg tacttcttgt ttaatgttta aagatactac 2640 ttattttaat gttatgtgta tggatgtttt atctatgtgt ttgtctgtat atagtgggca 2700 cgtactggtc tcagagccag aggaaggcat cagagtccct ggggttggaa ttaaagatgt 2760 ttgtgagtac ctgcgtgtat cctggacttc aaacccgggt cttcttcaag agcagccagt 2820 gctcttaacc actgaggatc tctccagcct catcgctgat ttaggaagga cttttactga 2880 tttggagtag ctgtaggcaa tgcagtctat gacgatttcc ttttagcagt tcttgtttgt 2940 tttcttaatg atagccatac tgattgctga gatttacagc agcactagca agctggaa 2998 13 1121 DNA Mus musculus misc_feature n=(a or c or t or g) 13 ctcgagtttt tttttttttt ttttggagaa gggnaacatt tattcattca acaaatnttg 60 atgacctgat ggggnagata actgagctag tcagcgcgta ggtagcaaac ataaggntat 120 agtaccccag ntaatggtct ncccacatgt cactgaagga gtgtcagttc tcagcatttt 180 acctttaatt ttaattttta cctctaaatg cgctttagga ggctacccac agttgatgac 240 aaacagtgta gccaggcatg ccagaactgt taccagcaga acttttggcc gactgtagct 300 ggcagtgttc tcagtagtgc agttcatgcc tggtgggtgt aactagggta caacgaagtc 360 actttgaact cttttgctaa ctaaataagc caaataaaca aatcatgaaa tactgattag 420 caatgcaata tttcatggca tgggaagagc ttcgacttct ccatcggtga caaggagcag 480 cttctggaag gaaggtctgg agaaaacaac tgacggggag ctccgaggag ccctgaacac 540 gtcactcaac agcactggcg ttgacacagc tgctgtggtc cagcagtcac tcagtggaga 600 gtgccaaagg gtgggcagac agncagncct acttcttcat ctccaggatg gcacttccag 660 gcccacggtt cttagcacta cagatgttgc agtattgtgc aggagcattc atgctcggca 720 taggcaggca ctccttgtgg aacatgtgcc ggcagtggaa gaccaccacg ctgaagggct 780 tcnctgcatc tgttgggagg atgggagaaa ggcatgattc acagatattc tcttcatcaa 840 ccagaacgcc tttcatttgg gttcggngca ttttttcaca caccaacgac aatgagtcag 900 ctacgaggat tttcttgcag ccttcccgaa gcagaatctt caagttataa tcttgcagaa 960 ttttaaccaa ggaatctctc aaattgggaa tctccattcc ttccttaatt cggtggataa 1020 gtagaatcgg gtccacatgt gtgccaatgt tgttcaacaa gccagtgata aatggtggtt 1080 tgtcgatgga gtatagaatc agatcttctc gtgccgaatt c 1121 14 779 DNA Mus musculus misc_feature n=(a or c or t or g) 14 ctcgagagat gccccacagt ccctcaggac ccgagtcagg taatctgcct ttggccttag 60 tgacctcctt ttctgggcga gtataccatc cactttcctc cctgacaggc agttcagtaa 120 cccaaccctt tcattcctcc ttcagttgtc aaagacaact taacatccaa gactaacaag 180 caagatgact caggagcatg gnctctgggt tcccctggca ccatgcatgg tgatgctagt 240 taaggctgac ttagctctta gcaaccttgg ttgggatagc ttaagctcat ctccactttc 300 ctaccaaaca gaaaagaatt tgagtcctct tgctatgagg ctctcgctcc catctcaggc 360 gagcttcctg cccctcaccc aagcttggga ggtagagtta tggagagggc aaggaagcag 420 gactggaaag atagacttat ggatccacca ctcataaagt cacaaagtcc cctcacacct 480 gctagactta gactctaaat cattacgttg tcaccaacag aggtgactcc tcaaccacaa 540 gagcctgtag tgagcttcaa gagagaagag gacaagncag acctggactg catgaccttg 600 cacctgtgat gaagtcacag caataggtga tgctcaaaaa gccccaataa aatgcaagac 660 agncaaacag aagccctgtc tgtccccatt ggtgggtaat gtagctgatg tggctggttc 720 tccttccttg acttcaccct gactatggga attgtccttc agtgcctcgt gccgaattc 779 15 981 DNA Mus musculus misc_feature n=(a or c or t or g) 15 ctcgagaggt gaaggcagaa gtatcacaag ttcaagttca aggncagcct gggcttcaca 60 agacccaaaa aaataaatat gaggncagtc caggctggga ctcaggtcac tgctgtgctg 120 agccatcgtc agagaagttt cttctttnnt tttgatagga gctaacacag cgacccacan 180 ctggacagnc tgcagtgagt gagtgagtaa gtgacctaaa agtgatgtct tcattaatct 240 cccctcccca ggcntcaggg agctctgagg aagaggaggc agaaagatgg tgagagccag 300 cagggatgga ggacaccaag gaagcagtgt cttccgacac aacaggactg gcatttagga 360 agtcacagag gctgtggctg cccagggcct gcacggtcca agctggctga gattccagtg 420 ctgagagaga caattcaaca cggnctccca cccctagnca agaagttatc tccaactgat 480 atccacttgc aaaggaaaaa attagggggn tagagagatg gctcagtggn taagagcact 540 gacttanaaa atagaaatng canattngnt nngangttng cnaaatngct gagaaatggc 600 caattggctg gaaaacttgc aacattgcct ggagaactgc caaattgcct ggagagctgc 660 caaattggcc tggagagctg cctacatggc ctggagagct gcccacatgg cctggagaac 720 tggctacatg tcctggagag ctgccaacat gtcctggaga tctgcctaca tggcctggag 780 aactgcctac atgacctgga gagctggcca catggcctgg agagctggct acatgacctg 840 gagagctgnc tacatggcct ggagagctgg ctacatggcc tggagagctg gctacatggc 900 ctggagagct ggctacatgg cctggagagc tggctacatg gcctggagag cctcccagca 960 aggcctctct aagccgaatt c 981 16 685 DNA Mus musculus misc_feature n=(a or c or t or g) 16 ctcgagatgc attaaagctt tgntgcagaa ggatccgagt gtgtcctgtg tgtgtgtcct 60 cactggcgag accctttatc acacagggac accccttagg ttggagtttt ccttgtaatg 120 tccactatac gtctgcttta tacaataata ttgnttaaat ttgnctctat catgaaatac 180 ctcactttcc ttatctgtat tgattgaaag ttttggtgga tgtaatagtt tgggcttgga 240 tctgaagtct tttagagttt attggacatg tgcctngatt cattggnttn aaaatcntcc 300 acnacttggg ggtgtaaagg ttacccacnc nattantgga ggttcttctg agttccagag 360 anaangantg agccaccngg aattctccct aaacacactt tgatcatttc ctgcctaacc 420 ctgcagagga aatattaata ccctgtagta ccaaaggaaa caaataagaa ggaagactgn 480 tctctcatgt ctggaggaag tttggtgaag gagtcttctg tttgctcaca taggagagat 540 ctaatacagc cactatccat aattaaaaat ctctgtgaga gaggcatgac gaggttctcc 600 cagtctgtca agggatgtga atatgtgttn ccctgtcatc ctgtcatgaa gcctctcttt 660 ctctctctct cctcgtgccg aattc 685 17 471 DNA Mus musculus misc_feature n=(a or c or t or g) 17 gaattcngcn ttggggtaca tggaccngga gagcttggnt acatggcctg gagagctggn 60 tacatggccc ggngagctgg tttnataaac ctggggangt tgggttnaat ggccccgggg 120 angtnggttn aatanaccng gggaggtgtc tgaaaanagt ggncacgtac tgttctcaga 180 cccagnggaa gncatcagag tcccctgggg ttggaattaa agatgtttgt gagtcnctgc 240 gtgtatcctg gacttcaaac ccgggtcttc ttcaagagca gccagtgctc ttaaccactg 300 agggatctct ccagcctcat cgctgattta ggaaggactt ttactgattt ggagtanctg 360 tagccaatnc agtctatgac gatttccttt tagcagttct tgtttgtttt cttaatgata 420 gccatactga ttgctgagat ttacagcagc actagcaagc tggaactcga g 471 18 467 DNA Mus musculus misc_feature n=(a or c or t or g) 18 ctcgagnttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 60 tttttnnnnn aanaaanttt taaagttttt ttttttttat naaaannttt ccaagggggg 120 gangggttag aaganagcca nagcctggnc ccccctgcca gaaaaaacca gaggggggtt 180 gatgtcccca agtccagttg tcaccctgaa gaagttcccc acgatttccc tggtggcccc 240 ccgggagtac gtccagagtg tcaccctttc catttgggag ctgtgggaag ggngtgggnt 300 ccctcccagn ggggccccaa acccttctcc tgaacagntc ctgatttctg accatctttc 360 caattccacg gattcaaaga gcatgaccct aggtaagcaa gccaggtcaa gagcattgct 420 tgtctgnagg aaaaggaagg gtccctcctg gcctcgtgcc gaattcc 467 19 13 PRT Mus musculus 19 Glu Leu Gln Arg Thr Ser Ser Val Ser Gly Pro Leu Ser 1 5 10 20 14 PRT Mus musculus 20 Phe Asn Ser Arg Arg Thr Ala Ser Asp His Ser Trp Ser Gly 1 5 10 21 13 PRT Mus musculus 21 Leu Arg Arg Lys Tyr Arg Ser Arg Glu Gln Pro Gln Ser 1 5 10 22 18 PRT Mus musculus 22 Ser Ala Gln Ser Leu Val Val Thr Leu Gly Arg Val Glu Gly Gly Ile 1 5 10 15 Arg Val 23 19 PRT Mus musculus 23 Cys Ser Ala Gln Ser Leu Val Val Thr Leu Gly Arg Val Glu Gly Gly 1 5 10 15 Ile Arg Val 24 17 PRT Mus musculus 24 Lys Ile Glu Gly Ser Ser Lys Cys Ala Pro Leu Arg Pro Ala Ser Arg 1 5 10 15 Leu 25 17 PRT Mus musculus 25 Cys Ala Pro Leu Arg Pro Ala Ser Arg Leu Pro Ala Ser Gln Thr Leu 1 5 10 15 Gly 26 16 PRT Mus musculus 26 Pro Pro Arg Glu Tyr Arg Ala Ser Gly Ser Arg Arg Gly Met Ala Tyr 1 5 10 15 27 17 PRT Mus musculus 27 Pro Pro Arg Glu Tyr Arg Ala Ser Gly Ser Arg Arg Gly Met Ala Tyr 1 5 10 15 Cys 28 16 PRT Mus musculus 28 Cys Lys Val Pro Arg Arg Arg Arg Thr Met Ala Asp Pro Asp Phe Trp 1 5 10 15 

What is claimed is:
 1. An antibody recognizing an antigen selected from the group consisting of peptide sequences SEQ ID NO:19 and SEQ ID NO:
 20. 